Professor of Pediatrics (Neonatology), of Microbiology and Immunology and, by courtesy, of Radiology and of Bioengineering

Pediatrics - Neonatal and Developmental Medicine

Bio

Bio

Dr. Contag, is a Professor in the Departments of Pediatrics, Radiology and Microbiology & Immunology at Stanford University, and a member of BioX Faculty for interdisciplinary sciences, and Immunology Faculty. Dr. Contag received his B.S. in Biology from the University of Minnesota, St. Paul in 1982. He received his Ph.D. in Microbiology from the University of Minnesota, Minneapolis in 1988 where he did his dissertation research under the direction of Professors Ashley Haase and Peter Plagemann on the topic of viral infections of the central nervous system. He was a postdoctoral fellow at Stanford University from 1990-1994 in the Department of Microbiology where he studied mother-to-infant transmission of HIV, and then joined the faculty in Pediatrics at Stanford in 1995 with a joint appointment in Microbiology and Immunology and a courtesy appointment in Radiology. Dr. Contag is the Associate Chief of Neonatal and Developmental Medicine, director of Stanford?s Center for Innovation in In Vivo Imaging (SCI3) and co-director of the Molecular Imaging Program at Stanford (MIPS). Dr. Contag is a pioneer in the field of molecular imaging and is developing imaging approaches aimed at revealing molecular processes in living subjects, including humans, and advancing therapeutic strategies through imaging. His laboratory develops macroscopic and microscopic optical imaging tools and uses imaging to assess tissue responses to stress, reveal immune cell migration patterns, understand stem cell biology and advance biological therapies. He is a founding member, and a past president of the Society for Molecular Imaging, and for his fundamental contributions in imaging, is a recipient of the Achievement Award from the Society for the Molecular Imaging. Dr. Contag is a Fellow of the World Molecular Imaging Society (WMIS) and currently President Elect of WMIS. The research mission of the Contag laboratory is to develop and use noninvasive imaging tools that can simultaneously reveal the nuances of biological processes and provide an overall picture of disease states for the purpose of developing and refining novel interventions. These imaging tools are sensitive and image over a range of scales from micro- to macroscopic, and are well-suited for the in vivo study of cellular and molecular biology. For the purpose of studying tumor biology in vivo, the Contag group is developing, and using, advanced microscopic tools with the aims of detecting and studying cancer at high resolution in vivo. These approaches use micro-optics to develop miniaturized cofocal microscopes and Raman endoscopes that can reach inside the body to interrogate disease states. This is enabling point-of-care microscopy that is changing the diagnostic paradigm from biopsy and histopathology to in vivo pathology. The opportunity to study tumor margins with arrays of microscopes will enable improved tumor detection and guided resections.

Research & Scholarship

Current Research and Scholarly Interests

Mammalian biology occurs in complex environments of living tissues and complex organ structures where there is potential for rapid change, and therefore we use multimodality imaging approaches to study the dynamics of biological processes. These strategies have cellular resolution and molecular specificity, and can reveal dynamic changes as they occur in the living body. We have developed imaging approaches based on optical reporter genes and have used them to reveal immune cell trafficking patterns, regulation of gene expression, extent of tumor growth, stem cell biology, and nature of host responses to infection. Our initial experimental approach was based on the observation that light can pass through mammalian tissues, much the same as when light from a flashlight is shined through one's hand in a dark room. The source of light in our approach is internal; that is, we use genes originating from fireflies and other "glow-in-the-dark" (bioluminescent) organisms to mark mammalian cells and pathogens. These labeled entities are then used in animal models of human biology and disease, and the light that they produce is externally monitored to reveal levels of expression, growth rate, or movement within tissue and organs. The strength of this method is that it can be used to simultaneously reveal the nuances of biological processes, and the overall biological response in living animals. Recently, we have revealed the kinetics of stem cell engraftment and hematopoietic reconstitution, elucidated the nature of minimal residual disease states following cancer therapy and identified tissue sites that pathogens use to evade the host immune response. Optical methods of molecular imaging are extremely powerful in preclinical models and have tremendous potential, but a wide range of tools is becoming available for studying biology in vivo. We therefore use many of these tools and approach biological questions with multimodality strategies. The focus of our efforts is the cells and molecules that control the body’s response to insult and enable regeneration of damaged tissues and organs.

Abstract

The detection of biomarker-targeting surface-enhanced Raman scattering (SERS) nanoparticles (NPs) in the human gastrointestinal tract has the potential to improve early cancer detection; however, a clinically relevant device with rapid Raman-imaging capability has not been described. Here we report the design and in vivo demonstration of a miniature, non-contact, opto-electro-mechanical Raman device as an accessory to clinical endoscopes that can provide multiplexed molecular data via a panel of SERS NPs. This device enables rapid circumferential scanning of topologically complex luminal surfaces of hollow organs (e.g., colon and esophagus) and produces quantitative images of the relative concentrations of SERS NPs that are present. Human and swine studies have demonstrated the speed and simplicity of this technique. This approach also offers unparalleled multiplexing capabilities by simultaneously detecting the unique spectral fingerprints of multiple SERS NPs. Therefore, this new screening strategy has the potential to improve diagnosis and to guide therapy by enabling sensitive quantitative molecular detection of small and otherwise hard-to-detect lesions in the context of white-light endoscopy.

Abstract

ABSTRACT. Topical application and quantification of targeted, surface-enhanced Raman scattering (SERS) nanoparticles offer a new technique that has the potential for early detection of epithelial cancers of hollow organs. Although less toxic than intravenous delivery, the additional washing required to remove unbound nanoparticles cannot necessarily eliminate nonspecific pooling. Therefore, we developed a real-time, ratiometric imaging technique to determine the relative concentrations of at least two spectrally unique nanoparticle types, where one serves as a nontargeted control. This approach improves the specific detection of bound, targeted nanoparticles by adjusting for working distance and for any nonspecific accumulation following washing. We engineered hardware and software to acquire SERS signals and ratios in real time and display them via a graphical user interface. We report quantitative, ratiometric imaging with nanoparticles at pM and sub-pM concentrations and at varying working distances, up to 50 mm. Additionally, we discuss optimization of a Raman endoscope by evaluating the effects of lens material and fiber coating on background noise, and theoretically modeling and simulating collection efficiency at various working distances. This work will enable the development of a clinically translatable, noncontact Raman endoscope capable of rapidly scanning large, topographically complex tissue surfaces for small and otherwise hard to detect lesions.

Abstract

Interactions among neighboring cells underpin many physiological processes ranging from early development to immune responses. When these interactions do not function properly, numerous pathologies, including infection and cancer, can result. Molecular imaging technologies, especially optical imaging, are uniquely suited to illuminate complex cellular interactions within the context of living tissues in the body. However, no tools yet exist that allow the detection of microscopic events, such as two cells coming into close proximity, on a global, whole-animal scale. We report here a broadly applicable, longitudinal strategy for probing interactions among cells in living subjects. This approach relies on the generation of bioluminescent light when two distinct cell populations come into close proximity, with the intensity of the optical signal correlating with relative cellular location. We demonstrate the ability of this reporter strategy to gauge cell-cell proximity in culture models in vitro and then evaluate this approach for imaging tumor-immune cell interactions using a murine breast cancer model. In these studies, our imaging strategy enabled the facile visualization of features that are otherwise difficult to observe with conventional imaging techniques, including detection of micrometastatic lesions and potential sites of tumor immunosurveillance. This proximity reporter will facilitate probing of numerous types of cell-cell interactions and will stimulate the development of similar techniques to detect rare events and pathological processes in live animals.

Abstract

Homeostatic erythropoiesis leads to the formation of mature red blood cells under non-stress conditions, and the production of new erythrocytes occurs as the need arises. In response to environmental stimuli, such as bone marrow transplantation, myelosuppression, or anemia, erythroid progenitors proliferate rapidly in a process referred to as stress erythropoiesis. We have previously demonstrated that heme oxygenase-1 (HO-1) deficiency leads to disrupted stress hematopoiesis. Here, we describe the specific effects of HO-1 deficiency on stress erythropoiesis.We used a transplant model to induce stress conditions. In irradiated recipients that received hmox(+/-) or hmox(+/+) bone marrow cells, we evaluated (i) the erythrocyte parameters in the peripheral blood; (ii) the staining intensity of CD71-, Ter119-, and CD49d-specific surface markers during erythroblast differentiation; (iii) the patterns of histological iron staining; and (iv) the number of Mac-1(+)-cells expressing TNF-?. In the spleens of mice that received hmox(+/-) cells, we show (i) decreases in the proerythroblast, basophilic, and polychromatophilic erythroblast populations; (ii) increases in the insoluble iron levels and decreases in the soluble iron levels; (iii) increased numbers of Mac-1(+)-cells expressing TNF-?; and (iv) decreased levels of CD49d expression in the basophilic and polychromatophilic erythroblast populations.As reflected by effects on secreted and cell surface proteins, HO-1 deletion likely affects stress erythropoiesis through the retention of erythroblasts in the erythroblastic islands of the spleen. Thus, HO-1 may serve as a therapeutic target for controlling erythropoiesis, and the dysregulation of HO-1 may be a predisposing condition for hematologic diseases.

Abstract

Targeted biological therapies hold tremendous potential for treatment of cancer, yet their use has been limited by constraints on delivery and effective tumor targeting. We combined an immune effector cell population [cytokine-induced killer (CIK) cells] with an oncolytic viral therapy to achieve directed delivery to, and regression of, tumors in both immunodeficient and immunocompetent mouse models. Preinfection of CIK cells with modified vaccinia virus resulted in a prolonged eclipse phase with the virus remaining hidden until interaction with the tumor. Whole-body imaging revealed that the cells retained their ability to traffic to and to infiltrate the tumor effectively before releasing the virus. These results illustrate the potential of combining biotherapeutics for synergistic effects that more effectively treat cancer.

Abstract

Hepatocellular carcinoma is generally refractory to clinical treatment. Here, we report that inactivation of the MYC oncogene is sufficient to induce sustained regression of invasive liver cancers. MYC inactivation resulted en masse in tumour cells differentiating into hepatocytes and biliary cells forming bile duct structures, and this was associated with rapid loss of expression of the tumour marker alpha-fetoprotein, the increase in expression of liver cell markers cytokeratin 8 and carcinoembryonic antigen, and in some cells the liver stem cell marker cytokeratin 19. Using in vivo bioluminescence imaging we found that many of these tumour cells remained dormant as long as MYC remain inactivated; however, MYC reactivation immediately restored their neoplastic features. Using array comparative genomic hybridization we confirmed that these dormant liver cells and the restored tumour retained the identical molecular signature and hence were clonally derived from the tumour cells. Our results show how oncogene inactivation may reverse tumorigenesis in the most clinically difficult cancers. Oncogene inactivation uncovers the pluripotent capacity of tumours to differentiate into normal cellular lineages and tissue structures, while retaining their latent potential to become cancerous, and hence existing in a state of tumour dormancy.

Abstract

The bacterium Listeria monocytogenes can cause a life-threatening systemic illness in humans. Despite decades of progress in animal models of listeriosis, much remains unknown about the processes of infection and colonization. Here, we report that L. monocytogenes can replicate in the murine gall bladder and provide evidence that its replication there is extracellular and intraluminal. In vivo bioluminescence imaging was employed to determine the location of the infection over time in live animals, revealing strong signals from the gall bladder over a period of several days, in diseased as well as asymptomatic animals. The data suggest that L. monocytogenes may be carried in the human gall bladder.

Abstract

To reveal the early events and dynamics of hematopoietic reconstitution in living animals in real-time, we used bioluminescence imaging to monitor engraftment from single luciferase-labeled hematopoietic stem cells (HSC) in irradiated recipients. Transplanted HSC generated discrete foci in the spleen and bone marrow (BM), at a frequency that correlated with BM compartment size. Initially detected foci could expand locally, seed other sites in BM or spleen, and/or recede with different kinetics. These studies reveal dynamic and variable patterns of engraftment from highly purified HSC and indicate that the final overall contribution of individual HSC to hematopoietic chimerism does not depend on the specific site of initial engraftment and expansion.

Abstract

Control of gene expression often involves an interwoven set of regulatory processes. As information regarding regulatory pathways may be lost in ex vivo analyses, we used bioluminescence to monitor gene expression in living mammals. Viral promoters fused to firefly luciferase as transgenes in mice allowed external monitoring of gene expression both superficially and in deep tissues. In vivo bioluminescence was detectable using either intensified or cooled charge-coupled device cameras, and could be detected following both topical and systemic delivery of substrate. In vivo control of the promoter from the human immunodeficiency virus was demonstrated. As a model for DNA-based therapies and vaccines, in vivo transfection of a luciferase expression vector (SV-40 promoter and enhancer controlling expression) was detected. We conclude that gene regulation, DNA delivery and expression can now be noninvasively monitored in living mammals using a luciferase reporter. Thus, real-time, noninvasive study of gene expression in living animal models for human development and disease is possible.

Abstract

The study of pathogenic processes is often limited to ex vivo assays and cell-culture correlates. A greater understanding of infectious diseases would be facilitated by in vivo analyses. Therefore, we have developed a method for detecting bacterial pathogens in a living host and used this method to evaluate disease processes for strains of Salmonella typhimurlum that differ in their virulence for mice. Three strains of Salmonella were marked with bioluminescence through transformation with a plasmid conferring constitutive expression of bacterial luciferase. Detection of photons transmitted through tissues of animals infected with bioluminescent Salmonella allowed localization of the bacteria to specific tissues. In this manner progressive infections were distinguished from those that were persistent or abortive. We observed patterns of bioluminescence that suggested the caecum may play a pivotal role in Salmonella pathogenesis. In vivo efficacy of an antibiotic was monitored using this optical method. This study demonstrates that real time non-invasive analyses of pathogenic events and pharmacological monitoring can be performed in vivo.

Abstract

We evaluated the small molecule coelenterazine as a potential reporter of cancer-associated superoxide anion in cell culture and in mice.The superoxide anion concentrations of various cancer cell lines were quantified by coelenterazine chemiluminescence in vitro. Coelenteramide fluorescence was detected via flow cytometry and fluorescent microscopy. Coelenterazine was used for the in vivo detection of cancer-associated superoxide anion using the 4T1 breast adenocarcinoma mouse model.Various cell lines in culture demonstrated different superoxide anion concentrations, with a signal range of 3.15?±?0.06 to 11.80?±?0.24 times that of background. In addition to chemiluminescent detection of coelenterazine, we demonstrated fluorescent detection of coelenteramide within the cytoplasm of cells. 4T1 murine mammary adenocarcinoma tumors in mice demonstrated significantly higher 2.13?±?0.19-fold coelenterazine-based chemiluminescence than that of surrounding normal tissues.Collectively, our results indicate that coelenterazine can be used to assay superoxide anion concentrations in cultured cancer cells and in tumors growing in mice.

Abstract

Small interfering RNAs (siRNAs) specifically and potently inhibit target gene expression. Pachyonychia congenita (PC) is a skin disorder caused by mutations in genes encoding keratin (K) 6a/b, K16, and K17, resulting in faulty intermediate filaments. A siRNA targeting a single nucleotide, PC-relevant mutation inhibits K6a expression and has been evaluated in the clinic with encouraging results.To better understand the pathophysiology of PC, and develop a model system to study siRNA delivery and visualize efficacy in skin, wild type (WT) and mutant K6a complementary DNAs (cDNAs) were fused to either enhanced green fluorescent protein or tandem tomato fluorescent protein cDNA to allow covisualization of mutant and WT K6a expression in mouse footpad skin using a dual fluorescence in vivo confocal imaging system equipped with 488 and 532 nm lasers.Expression of mutant K6a/reporter resulted in visualization of keratin aggregates, while expression of WT K6a/reporter led to incorporation into filaments. Addition of mutant K6a-specific siRNA resulted in inhibition of mutant, but not WT, K6a/reporter expression.Intravital imaging offers subcellular resolution for tracking functional activity of siRNA in real time and enables detailed analyses of therapeutic effects in individual mice to facilitate development of nucleic acid-based therapeutics for skin disorders.

A role of the adaptive immune system in glucose homeostasis.BMJ open diabetes research & careBronsart, L. L., Contag, C. H.2016; 4 (1)

Abstract

The immune system, including the adaptive immune response, has recently been recognized as having a significant role in diet-induced insulin resistance. In this study, we aimed to determine if the adaptive immune system also functions in maintaining physiological glucose homeostasis in the absence of diet-induced disease.SCID mice and immunocompetent control animals were phenotypically assessed for variations in metabolic parameters and cytokine profiles. Additionally, the glucose tolerance of SCID and immunocompetent control animals was assessed following introduction of a high-fat diet.SCID mice on a normal chow diet were significantly insulin resistant relative to control animals despite having less fat mass. This was associated with a significant increase in the innate immunity-stimulating cytokines granulocyte colony-stimulating factor, monocyte chemoattractant protein 1 (MCP1), and MCP3. Additionally, the SCID mouse phenotype was exacerbated in response to a high-fat diet as evidenced by the further significant progression of glucose intolerance.These results support the notion that the adaptive immune system plays a fundamental biological role in glucose homeostasis, and that the absence of functional B and T cells results in disruption in the concentrations of various cytokines associated with macrophage proliferation and recruitment. Additionally, the absence of functional B and T cells is not protective against diet-induced pathology.

Abstract

Monogenic skin diseases arise from well-defined single gene mutations, and in some cases a single point mutation. As the target cells are superficial, these diseases are ideally suited for treatment by nucleic acid-based therapies as well as monitoring through a variety of noninvasive imaging technologies. Despite the accessibility of the skin, there remain formidable barriers for functional delivery of nucleic acids to the target cells within the dermis and epidermis. These barriers include the stratum corneum and the layered structure of the skin, as well as more locally, the cellular, endosomal and nuclear membranes. A wide range of technologies for traversing these barriers has been described and moderate success has been reported for several approaches. The lessons learned from these studies include the need for combinations of approaches to facilitate nucleic acid delivery across these skin barriers and then functional delivery across the cellular and nuclear membranes for expression (e.g., reporter genes, DNA oligonucleotides or shRNA) or into the cytoplasm for regulation (e.g., siRNA, miRNA, antisense oligos). The tools for topical delivery that have been evaluated include chemical, physical and electrical methods, and the development and testing of each of these approaches has been greatly enabled by imaging tools. These techniques allow delivery and real time monitoring of reporter genes, therapeutic nucleic acids and also triplex nucleic acids for gene editing. Optical imaging is comprised of a number of modalities based on properties of light-tissue interaction (e.g., scattering, autofluorescence, and reflectance), the interaction of light with specific molecules (e.g., absorbtion, fluorescence), or enzymatic reactions that produce light (bioluminescence). Optical imaging technologies operate over a range of scales from macroscopic to microscopic and if necessary, nanoscopic, and thus can be used to assess nucleic acid delivery to organs, regions, cells and even subcellular structures. Here we describe the animal models, reporter genes, imaging approaches and general strategies for delivery of nucleic acids to cells in the skin for local expression (e.g., plasmid DNA) or gene silencing (e.g., siRNA) with the intent of developing nucleic acid-based therapies to treat diseases of the skin.

Abstract

Here, we evaluate [(99m)Tc]annexin V-128, an in vivo marker of apoptosis, for single photon emission computed tomography (SPECT) imaging of localization and antibiotic treatment of disseminated bacterial infection, using a well-described mouse model that employs bioluminescent Listeria monocytogenes.Sixteen groups of five mice in six separate experiments were infected with bioluminescent Listeria, and in vivo bioluminescence imaging (BLI) was performed each day, to assess the location and extent of infection and response to antibiotics. [(99m)Tc]annexin V-128 was then injected for SPECT imaging, and the two sets of images were correlated and validated.Signals from BLI and [(99m)Tc]annexin V-128 SPECT co-localized within the spleen and other organs including bone marrow, intestine, nasopharynx, and brain. Decreases in [(99m)Tc]annexin V-128 uptake and BLI signal within the spleen directly reflected the reduction of bacterial infection by ampicillin treatment.Tc-99m-Annexin V-128 uptake as observed by SPECT allowed for the detection of systemic listeriosis and ampicillin treatment in mice. [(99m)Tc]annexin V-128 should be further explored for the assessment of bacterial spread and antibiotic efficacy in patients with disseminated bacterial infection.

Abstract

Malignant neoplastic lesions derived from epithelial tissue, carcinomas, account for 80% to 100% of all human cancers including some of the most deadly diseases such as cervical and non-small cell lung cancer. Many of these carcinomas present at readily accessible epithelial surfaces offering unique detection opportunities. Effective clinical management of carcinomas is enabled by early detection, at a time when full surgical resection is possible and before invasion of adjacent tissue or significant intravasation into blood vessels leading to metastasis. Good prognosis with long-term disease-free survival is more likely after early detection when progression is limited. At present, detection of carcinomas at epithelial surfaces largely relies on routine inspection with the naked eye (e.g., skin and oropharynx) or simple white light tools (e.g., cervix and colon). Emerging optical tools based on differential refraction, absorption, reflection, scattering, or fluorescence of carcinomas relative to normal tissues enable label-free visualization of neoplasia. However, the differences in intrinsic optical properties of normal and malignant tissues can be subtle, and relying on these may lead to high miss rates. Enhanced optical contrast offered by molecularly targeted agents can be used to improve early detection; and given that optical imaging and sensing tools can be readily combined, integrated systems that image over a range of scales, or detect multiple parameters, can be developed to aid in early detection. Diagnosis is, at present, made by histologic examination of tissue biopsies after identification of suspicious lesions. Miniature and handheld microscopic imaging tools have recently been developed, and integration of these tools with wide-field optical surveillance devices offers both rapid detection and confirmatory histologic examination at the point-of-care, that can provide guidance for biopsy and/or resection. A wide variety of targeted probe strategies have been described with demonstrated benefit in preclinical models and in a limited number of human studies. Here, we present examples of integrated multimodality optical imaging and sensing tools that use combinations of intrinsic and extrinsic optical contrast for early detection or margin delineation for carcinomas at epithelial surfaces. We will discuss several new technologies that have use in detecting the most common carcinomas that derive from the epithelium of the skin, gastrointestinal and urogenital tracts, and bronchoalveoli.

Abstract

Extracellular vesicles (EVs), specifically exosomes and microvesicles (MVs), are presumed to play key roles in cell-cell communication via transfer of biomolecules between cells. The biogenesis of these two types of EVs differs as they originate from either the endosomal (exosomes) or plasma (MVs) membranes. To elucidate the primary means through which EVs mediate intercellular communication, we characterized their ability to encapsulate and deliver different types of macromolecules from transiently transfected cells. Both EV types encapsulated reporter proteins and mRNA but only MVs transferred the reporter function to recipient cells. De novo reporter protein expression in recipient cells resulted only from plasmid DNA (pDNA) after delivery via MVs. Reporter mRNA was delivered to recipient cells by both EV types, but was rapidly degraded without being translated. MVs also mediated delivery of functional pDNA encoding Cre recombinase in vivo to tissues in transgenic Cre-lox reporter mice. Within the parameters of this study, MVs delivered functional pDNA, but not RNA, whereas exosomes from the same source did not deliver functional nucleic acids. These results have significant implications for understanding the role of EVs in cellular communication and for development of EVs as delivery tools. Moreover, studies using EVs from transiently transfected cells may be confounded by a predominance of pDNA transfer.

Abstract

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Abstract

Atherosclerosis is a major cause of global morbidity and mortality that could benefit from novel targeted therapeutics. Recent studies have shown efficient and local drug delivery with nanoparticles, although the nanoparticle targeting mechanism for atherosclerosis has not yet been fully elucidated. Here we used in vivo and ex vivo multimodal imaging to examine permeability of the vessel wall and atherosclerotic plaque accumulation of fluorescently labeled liposomal nanoparticles in a rabbit model. We found a strong correlation between permeability as established by in vivo dynamic contrast enhanced magnetic resonance imaging and nanoparticle plaque accumulation with subsequent nanoparticle distribution throughout the vessel wall. These key observations will enable the development of nanotherapeutic strategies for atherosclerosis.

Abstract

A method for non-invasive visualization of genetically labeled cells in animal disease models with micrometer-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the 'optical window' above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three new red-excitable monomeric FPs obtained by structure-guided mutagenesis of mNeptune. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence than mNeptune, whereas the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness. We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts into myocytes in living mice with high anatomical detail.

Abstract

The promise of cell-based immunotherapies for the treatment of cancer offers the potential of therapeutic synergy with chemo- and radiotherapies that may overcome current limitations leading to durable responses and prevention of recurrence. There is a wide array of cell-based immunotherapies that are either poised to enter cancer clinical trials or are in clinical trials, and many are showing some success. Yet within this field, there are clear obstacles that need to be overcome, including limited access across tissue barriers, development of antigen tolerance, and the immunosuppressive microenvironment of tumors. Through an understanding of immune cell signaling and trafficking, immune cell populations can be selected for adoptive transfer, and delivery strategies can be developed that circumvent these obstacles to effectively direct populations of cells with robust anti-tumor efficacy to the target. Within the realm of immune cell therapies, cytokine-induced killer (CIK) cells have demonstrated promising trafficking patterns, effective delivery of synergistic therapeutics, and stand-alone efficacy. Here, we discuss the next generation of CIK therapies and their application for the effective treatment of a wide variety of cancers.

Abstract

Advances in noninvasive imaging technologies that allow for in vivo dynamic monitoring of cells and cellular function in living research subjects have revealed new insights into cell biology in the context of intact organs and their native environment. In the field of hematopoiesis and stem cell research, studies of cell trafficking involved in injury repair and hematopoietic engraftment have made great progress using these new tools. Stem cells present unique challenges for imaging since after transplantation, they proliferate dramatically and differentiate. Therefore, the imaging modality used needs to have a large dynamic range, and the genetic regulatory elements used need to be stably expressed during differentiation. Multiple imaging technologies using different modalities are available, and each varies in sensitivity, ease of data acquisition, signal to noise ratios (SNR), substrate availability, and other parameters that affect utility for monitoring cell fates and function. For a given application, there may be several different approaches that can be used. For mouse models, clinically validated technologies such as magnetic resonance imaging (MRI) and positron emission tomography (PET) have been joined by optical imaging techniques such as in vivo bioluminescence imaging (BLI) and fluorescence imaging (FLI), and all have been used to monitor bone marrow and stem cells after transplantation into mice. Photoacoustic imaging that utilizes the sound created by the thermal expansion of absorbed light to generate an image best represents hybrid technologies. Each modality requires that the cells of interest be marked with a genetic reporter that acts as a label making them uniquely visible using that technology. For each modality, there are several labels to choose from. Multiple methods for applying these different labels are available. This chapter provides an overview of the imaging technologies and commonly used labels for each, as well as detailed protocols for gene delivery into hematopoietic cells for the purposes of applying these specific labels to cell trafficking. The goal of this chapter is to provide adequate background information to allow the design and implementation of an experimental system for in vivo imaging in mice.

Abstract

Abstract Hypoxia-inducible factor-1 alpha (HIF-1?) gene therapy holds great promise for the treatment of myocardial ischemia. Both preclinical and clinical evaluations of this therapy are underway and can benefit from a vector strategy that allows noninvasive assessment of HIF-1? expression as an objective measure of gene delivery. We have developed a novel bidirectional plasmid vector (pcTnT-HIF-1?-VP2-TSTA-fluc), which employs the cardiac troponin T (cTnT) promoter in conjunction with a two-step transcriptional amplification (TSTA) system to drive the linked expression of a recombinant HIF-1? gene (HIF-1?-VP2) and the firefly luciferase gene (fluc). The firefly luciferase (FLuc) activity serves as a surrogate for HIF-1?-VP2 expression, and can be noninvasively assessed in mice using bioluminescence imaging after vector delivery. Transfection of cultured HL-1 cardiomyocytes with pcTnT-HIF-1?-VP2-TSTA-fluc led to a strong correlation between FLuc and HIF-1?-dependent vascular endothelial growth factor expression (r(2)=0.88). Intramyocardial delivery of pcTnT-HIF-1?-VP2-TSTA-fluc into infarcted mouse myocardium led to persistent HIF-1?-VP2 expression for 4 weeks, even though it improved neither CD31+ microvessel density nor echocardiographically determined left ventricular systolic function. These results lend support to recent findings of suboptimal efficacy associated with plasmid-mediated HIF-1? therapy. The imaging techniques developed herein should be useful for further optimizing HIF-1?-VP2 therapy in preclinical models of myocardial ischemia.

Abstract

We previously identified short synthetic shRNAs (sshRNAs) that target a conserved hepatitis C virus (HCV) sequence within the internal ribosome entry site (IRES) of HCV and potently inhibit HCV IRES-linked gene expression. To assess in vivo liver delivery and activity, the HCV-directed sshRNA SG220 was formulated into lipid nanoparticles (LNP) and injected i.v. into mice whose livers supported stable HCV IRES-luciferase expression from a liver-specific promoter. After a single injection, RNase protection assays for the sshRNA and (3)H labeling of a lipid component of the nanoparticles showed efficient liver uptake of both components and long-lasting survival of a significant fraction of the sshRNA in the liver. In vivo imaging showed a dose-dependent inhibition of luciferase expression (>90% 1 day after injection of 2.5?mg/kg sshRNA) with t1/2 for recovery of about 3 weeks. These results demonstrate the ability of moderate levels of i.v.-injected, LNP-formulated sshRNAs to be taken up by liver hepatocytes at a level sufficient to substantially suppress gene expression. Suppression is rapid and durable, suggesting that sshRNAs may have promise as therapeutic agents for liver indications.Molecular Therapy-Nucleic Acids (2013) 2, e123; doi:10.1038/mtna.2013.50; published online 17 September 2013.

Abstract

Endoscopic imaging is an invaluable diagnostic tool allowing minimally invasive access to tissues deep within the body. It has played a key role in screening colon cancer and is credited with preventing deaths through the detection and removal of precancerous polyps. However, conventional white-light endoscopy offers physicians structural information without the biochemical information that would be advantageous for early detection and is essential for molecular typing. To address this unmet need, we have developed a unique accessory, noncontact, fiber optic-based Raman spectroscopy device that has the potential to provide real-time, multiplexed functional information during routine endoscopy. This device is ideally suited for detection of functionalized surface-enhanced Raman scattering (SERS) nanoparticles as molecular imaging contrast agents. This device was designed for insertion through a clinical endoscope and has the potential to detect and quantify the presence of a multiplexed panel of tumor-targeting SERS nanoparticles. Characterization of the Raman instrument was performed with SERS particles on excised human tissue samples, and it has shown unsurpassed sensitivity and multiplexing capabilities, detecting 326-fM concentrations of SERS nanoparticles and unmixing 10 variations of colocalized SERS nanoparticles. Another unique feature of our noncontact Raman endoscope is that it has been designed for efficient use over a wide range of working distances from 1 to 10 mm. This is necessary to accommodate for imperfect centering during endoscopy and the nonuniform surface topology of human tissue. Using this endoscope as a key part of a multiplexed detection approach could allow endoscopists to distinguish between normal and precancerous tissues rapidly and to identify flat lesions that are otherwise missed.

Abstract

The development of siRNA-based gene silencing therapies has significant potential for effectively treating debilitating genetic, hyper-proliferative or malignant skin conditions caused by aberrant gene expression. To be efficacious and widely accepted by physicians and patients, therapeutic siRNAs must access the viable skin layers in a stable and functional form, preferably without painful administration. In this study we explore the use of minimally-invasive steel microneedle devices to effectively deliver siRNA into skin. A simple, yet precise microneedle coating method permitted reproducible loading of siRNA onto individual microneedles. Following recovery from the microneedle surface, lamin A/C siRNA retained full activity, as demonstrated by significant reduction in lamin A/C mRNA levels and reduced lamin A/C protein in HaCaT keratinocyte cells. However, lamin A/C siRNA pre-complexed with a commercial lipid-based transfection reagent (siRNA lipoplex) was less functional following microneedle coating. As Accell-modified "self-delivery" siRNA targeted against CD44 also retained functionality after microneedle coating, this form of siRNA was used in subsequent in vivo studies, where gene silencing was determined in a transgenic reporter mouse skin model. Self-delivery siRNA targeting the reporter (luciferase/GFP) gene was coated onto microneedles and delivered to mouse footpad. Quantification of reporter mRNA and intravital imaging of reporter expression in the outer skin layers confirmed functional in vivo gene silencing following microneedle delivery of siRNA. The use of coated metal microneedles represents a new, simple, minimally-invasive, patient-friendly and potentially self-administrable method for the delivery of therapeutic nucleic acids to the skin.

Abstract

Despite the development of a large number of promising candidates, few contrast agents for established medical imaging modalities have successfully been translated over the past decade. The emergence of new imaging contrast agents that employ biomedical optics is further complicated by the relative infancy of the field and the lack of approved imaging devices compared to more established clinical modalities such as nuclear medicine. Herein, we propose a navigational approach (as opposed to a fixed "roadmap") for translation of optical imaging agents that is (i) proposed through consensus by four academic research programs that are part of the cooperative U54 NCI Network for Translational Research, (ii) developed through early experiences for translating optical imaging agents in order to meet distinctly varied needs in cancer diagnostics, and (iii) adaptable to the rapidly changing environment of academic medicine. We describe the pathways by which optical imaging agents are synthesized, qualified, and validated for preclinical testing, and ultimately translated for "first-in-humans" studies using investigational optical imaging devices. By identifying and adopting consensus approaches for seemingly disparate optical imaging modalities and clinical indications, we seek to establish a systematic method for navigating the ever-changing "roadmap" to most efficiently arrive at the destination of clinical adoption and improved outcome and survivorship for cancer patients.

Abstract

Despite the development of potent siRNAs that effectively target genes responsible for skin disorders, translation to the clinic has been hampered by inefficient delivery through the stratum corneum barrier and into the live cells of the epidermis. Although hypodermic needles can be used to transport siRNA through the stratum corneum, this approach is limited by pain caused by the injection and the small volume of tissue that can be accessed by each injection. The use of microneedle arrays is a less painful method for siRNA delivery, but restricted payload capacity limits this approach to highly potent molecules. To address these challenges, a commercially available motorized microneedle array skin delivery device was evaluated. This device combines the positive elements of both hypodermic needles and microneedle array technologies with little or no pain to the patient. Application of fluorescently tagged self-delivery (sd)-siRNA to both human and murine skin resulted in distribution throughout the treated skin. In addition, efficient silencing (78% average reduction) of reporter gene expression was achieved in a transgenic fluorescent reporter mouse skin model. These results indicate that this device effectively delivers functional sd-siRNA with an efficiency that predicts successful clinical translation.Molecular Therapy-Nucleic Acids (2013) 2, e129; doi:10.1038/mtna.2013.56; published online 22 October 2013.

Abstract

Miniature microscopes are being developed to examine tissue in situ for early anatomic and molecular indicators of disease, in real time, and at cellular resolution. These new devices will lead to a shift from the current diagnostic paradigm of biopsy followed by histopathology and recommended therapy, to one of non-invasive point-of-care diagnosis with the possibility of treatment in the same session. This potential revolution in disease management may have a major impact on the training of future physicians to include the use and interpretation of real-time in vivo microscopic data, and will also affect the emerging fields of telepathology and telemedicine. Implementation of new technologies into clinical practice is a complex process that requires multidisciplinary communication and collaboration among clinicians, engineers and scientists. As such, our aim is to provide a forward-looking view of the critical issues facing the development of new technologies and directing clinical education. Here, we focus on the use of in vivo microscopy for detection of malignant and pre-malignant lesions as well as for guiding therapy. We will highlight some of the areas in which in vivo microscopy could address unmet clinical needs, and then review the technological challenges that are being addressed, or need to be addressed, for in vivo microscopy to become an effective clinical tool.

Abstract

Although mechanical osteotomies are frequently made on the craniofacial skeleton, collateral thermal, and mechanical trauma to adjacent bone tissue causes cell death and may delay healing. The present study evaluated the use of plasma-mediated laser ablation using a femtosecond laser to circumvent thermal damage and improve bone regeneration.Critical-size circular calvarial defects were created with a trephine drill bit or with a Ti:Sapphire femtosecond pulsed laser. Healing was followed using micro-CT scans for 8 weeks. Calvaria were also harvested at various time points for histological analysis. Finally, scanning electron microscopy was used to analyze the microstructure of bone tissue treated with the Ti:Sapphire laser, and compared to that treated with the trephine bur.Laser-created defects healed significantly faster than those created mechanically at 2, 4, and 6 weeks post-surgery. However, at 8 weeks post-surgery, there was no significant difference. In the drill osteotomy treatment group, empty osteocyte lacunae were seen to extend 699?±?27?µm away from the edge of the defect. In marked contrast, empty osteocyte lacunae were seen to extend only 182?±?22?µm away from the edge of the laser-created craters. Significantly less ossification and formation of irregular woven bone was noted on histological analysis for drill defects.We demonstrate accelerated bone healing after femtosecond laser ablation in a calvarial defect model compared to traditional mechanical drilling techniques. Improved rates of early regeneration make plasma-mediated ablation of the craniofacial skeleton advantageous for applications to osteotomy.

Abstract

The unambiguous demarcation of tumor margins is critical at the final stages in the surgical treatment of brain tumors because patient outcomes have been shown to correlate with the extent of resection. Real-time high-resolution imaging with the aid of a tumor-targeting fluorescent contrast agent has the potential to enable intraoperative differentiation of tumor versus normal tissues with accuracy approaching the current gold standard of histopathology. In this study, a monoclonal antibody targeting the vascular endothelial growth factor receptor 1 (VEGFR-1) was conjugated to fluorophores and evaluated as a tumor contrast agent in a transgenic mouse model of medulloblastoma. The probe was administered topically, and its efficacy as an imaging agent was evaluated in vitro using flow cytometry, as well as ex vivo on fixed and fresh tissues through immunohistochemistry and dual-axis confocal microscopy, respectively. Results show a preferential binding to tumor versus normal tissue, suggesting that a topically applied VEGFR-1 probe can potentially be used with real-time intraoperative optical sectioning microscopy to guide brain tumor resections.

Abstract

Development of RNA interference (RNAi)-based therapeutics has been hampered by the lack of effective and efficient means of delivery. Reliable model systems for screening and optimizing delivery of RNAi-based agents in vivo are crucial for preclinical research aimed at advancing nucleic acid-based therapies. We describe here a dual fluorescent reporter xenograft melanoma model prepared by intradermal injection of human A375 melanoma cells expressing tandem tomato fluorescent protein (tdTFP) containing a small interfering RNA (siRNA) target site as well as enhanced green fluorescent protein (EGFP), which is used as a normalization control. Intratumoral injection of a siRNA specific to the incorporated siRNA target site, complexed with a cationic lipid that has been optimized for in vivo delivery, resulted in 65%±11% knockdown of tdTFP relative to EGFP quantified by in vivo imaging and 68%±10% by reverse transcription-quantitative polymerase chain reaction. No effect was observed with nonspecific control siRNA treatment. This model provides a platform on which siRNA delivery technologies can be screened and optimized in vivo.

Abstract

Beneficial medical laser ablation removes material efficiently with minimal collateral damage. A Mark-III free electron laser (FEL), at a wavelength of 6.45 ?m has demonstrated minimal damage and high ablation yield in ocular and neural tissues. While this wavelength has shown promise for surgical applications, further advances are limited by the high overhead for FEL use. Alternative mid-infrared sources are needed for further development. We compared the FEL with a 5-?s pulse duration with a Q-switched ZGP-OPO with a 100-ns pulse duration at mid-infrared wavelengths. There were no differences in the ablation threshold of water and mouse dermis with these two sources in spite of the difference in their pulse structures. There was a significant difference in crater depth between the ZGP:OPO and the FEL. At 6.1 ?m, the OPO craters are eight times the depth of the FEL craters. The OPO craters at 6.45 and 6.73 ?m were six and five times the depth of the FEL craters, respectively. Bright-field (pump-probe) images showed the classic ablation mechanism from formation of a plume through collapse and recoil. The crater formation, ejection, and collapse phases occurred on a faster time-scale with the OPO than with the FEL. This research showed that a ZGP-OPO laser could be a viable alternative to FEL for clinical applications.

Abstract

Full understanding of the biological significance of negative feedback processes requires interrogation at multiple scales as follows: in single cells, cell populations, and live animals in vivo. The transcriptionally coupled I?B?/NF-?B negative feedback loop, a pivotal regulatory node of innate immunity and inflammation, represents a model system for multiscalar reporters. Using a ?B(5)?I?B?-FLuc bioluminescent reporter, we rigorously evaluated the dynamics of ??B? degradation and subsequent NF-?B transcriptional activity in response to diverse modes of TNF? stimulation. Modulating TNF? concentration or pulse duration yielded complex, reproducible, and differential ??B? dynamics in both cell populations and live single cells. Tremendous heterogeneity in the transcriptional amplitudes of individual responding cells was observed, which was greater than the heterogeneity in the transcriptional kinetics of responsive cells. Furthermore, administration of various TNF? doses in vivo generated ??B? dynamic profiles in the liver resembling those observed in single cells and populations of cells stimulated with TNF? pulses. This suggested that dose modulation of circulating TNF? was perceived by hepatocytes in vivo as pulses of increasing duration. Thus, a robust bioluminescent reporter strategy enabled rigorous quantitation of NF-?B/??B? dynamics in both live single cells and cell populations and furthermore, revealed reproducible behaviors that informed interpretation of in vivo studies.

Abstract

Treatment of skin disorders with short interfering RNA (siRNA)-based therapeutics requires the development of effective delivery methodologies that reach target cells in affected tissues. Successful delivery of functional siRNA to the epidermis requires (1) crossing the stratum corneum, (2) transfer across the keratinocyte membrane, followed by (3) incorporation into the RNA-induced silencing complex. We have previously demonstrated that treatment with microneedle arrays loaded with self-delivery siRNA (sd-siRNA) can achieve inhibition of reporter gene expression in a transgenic mouse model. Furthermore, treatment of human cultured epidermal equivalents with sd-siRNA resulted in inhibition of target gene expression. Here, we demonstrate inhibition of CD44, a gene that is uniformly expressed throughout the epidermis, by sd-siRNA both in vitro (cultured human epidermal skin equivalents) and in vivo (full-thickness human skin equivalents xenografted on immunocompromised mice). Treatment of human skin equivalents with CD44 sd-siRNA markedly decreased CD44 mRNA levels, which led to a reduction of the target protein as confirmed by immunodetection in epidermal equivalent sections with a CD44-specific antibody. Taken together, these results demonstrate that sd-siRNA, delivered by microneedle arrays, can reduce expression of a targeted endogenous gene in a human skin xenograft model.

Abstract

High-resolution image guidance for resection of residual tumor cells would enable more precise and complete excision for more effective treatment of cancers, such as medulloblastoma, the most common pediatric brain cancer. Numerous studies have shown that brain tumor patient outcomes correlate with the precision of resection. To enable guided resection with molecular specificity and cellular resolution, molecular probes that effectively delineate brain tumor boundaries are essential. Therefore, we developed a bioinformatics approach to analyze micro-array datasets for the identification of transcripts that encode candidate cell surface biomarkers that are highly enriched in medulloblastoma. The results identified 380 genes with greater than a two-fold increase in the expression in the medulloblastoma compared with that in the normal cerebellum. To enrich for targets with accessibility for extracellular molecular probes, we further refined this list by filtering it with gene ontology to identify genes with protein localization on, or within, the plasma membrane. To validate this meta-analysis, the top 10 candidates were evaluated with immunohistochemistry. We identified two targets, fibrillin 2 and EphA3, which specifically stain medulloblastoma. These results demonstrate a novel bioinformatics approach that successfully identified cell surface and extracellular candidate markers enriched in medulloblastoma versus adjacent cerebellum. These two proteins are high-value targets for the development of tumor-specific probes in medulloblastoma. This bioinformatics method has broad utility for the identification of accessible molecular targets in a variety of cancers and will enable probe development for guided resection.

Abstract

Listeriosis is one of the most lethal bacterial diseases for fetuses and infants. However, pregnant women who get infected with Listeria may experience only mild symptoms, making the diagnosis difficult, even when the fetus is fatally infected.To reveal features of this infection, we conducted a multimodality imaging study of Listeria-induced miscarriage, using a pregnant mouse model. In this model, fetal morbidity and mortality can be observed in utero, noninvasively, and the timing and extent of infection can be carefully controlled. By employing in vivo bioluminescence imaging (BLI), perinatal infections were localized over time such that a correlation of infection to outcome could be determined without the need to kill the animal subject. The morbidity and viability of fetuses were assessed with ultrasound, and fetal morphology was imaged using magnetic resonance imaging (MRI).The ultrasound revealed sustained fetal bradycardia, the slowing of the fetal heartbeat, in infected fetuses, with an association between slowed fetal heart rate and strong bioluminescent signal.Uninfected fetuses showing no bioluminescent signal in the same uterine horn exhibited normal heartbeats. Thus, fetal bradycardia during infection was localized to the infected fetus and was not systemic or disseminated.

Abstract

Celastrol, an active compound extracted from the root of the Chinese medicine "Thunder of God Vine" (Tripterygium wilfordii), exhibits anticancer, antioxidant and anti-inflammatory activities, and interest in the therapeutic potential of celastrol is increasing. However, described side effects following treatment are significant and require investigation prior to initiating clinical trials. Here, we investigated the effects of celastrol on the adult murine hematopoietic system.Animals were treated daily with celastrol over a four-day period and peripheral blood, bone marrow, spleen, and peritoneal cavity were harvested for cell phenotyping. Treated mice showed specific impairment of the development of B cells and erythrocytes in all tested organs. In bone marrow, these alterations were accompanied by decreases in populations of common lymphoid progenitors (CLP), common myeloid progenitors (CMP) and megakaryocyte-erythrocyte progenitors (MEP).These results indicate that celastrol acts through regulators of adult hematopoiesis and could be used as a modulator of the hematopoietic system. These observations provide valuable information for further assessment prior to clinical trials.

Abstract

Medical imaging is an invaluable tool for diagnosis, surgical guidance, and assessment of treatment efficacy. The Network for Translational Research (NTR) for Optical Imaging consists of four research groups working to "bridge the gap" between lab discovery and clinical use of fluorescence- and photoacoustic-based imaging devices used with imaging biomarkers. While the groups are using different modalities, all the groups face similar challenges when attempting to validate these systems for FDA approval and, ultimately, clinical use. Validation steps taken, as well as future needs, are described here. The group hopes to provide translational validation guidance for itself, as well as other researchers.

Abstract

Near-infrared confocal microendoscopy is a promising technique for deep in vivo imaging of tissues and can generate high-resolution cross-sectional images at the micron-scale. We demonstrate the use of a dual-axis confocal (DAC) near-infrared fluorescence microendoscope with a 5.5-mm outer diameter for obtaining clinical images of human colorectal mucosa. High-speed two-dimensional en face scanning was achieved through a microelectromechanical systems (MEMS) scanner while a micromotor was used for adjusting the axial focus. In vivo images of human patients are collected at 5 frames/sec with a field of view of 362×212 ?m(2) and a maximum imaging depth of 140 ?m. During routine endoscopy, indocyanine green (ICG) was topically applied a nonspecific optical contrasting agent to regions of the human colon. The DAC microendoscope was then used to obtain microanatomic images of the mucosa by detecting near-infrared fluorescence from ICG. These results suggest that DAC microendoscopy may have utility for visualizing the anatomical and, perhaps, functional changes associated with colorectal pathology for the early detection of colorectal cancer.

Abstract

Small interfering RNA (siRNA) is a highly potent drug in gene-based therapy with a challenge of being delivered in a sustained manner. Nanoparticle drug delivery systems allow for incorporating and controlled release of therapeutic payloads. We demonstrate that solid lipid nanoparticles can incorporate and provide sustained release of siRNA. Tristearin solid lipid nanoparticles, made by nanoprecipitation, were loaded with siRNA (4.4-5.5 wt % loading ratio) using a hydrophobic ion pairing approach that employs the cationic lipid DOTAP. Intradermal injection of these nanocarriers in mouse footpads resulted in prolonged siRNA release over a period of 10-13 days. In vitro cell studies showed that the released siRNA retained its activity. Nanoparticles developed in this study offer an alternative approach to polymeric nanoparticles for encapsulation and sustained delivery of siRNA with the advantage of being prepared from physiologically well-tolerated materials.

Abstract

The accessibility of skin makes it an ideal target organ for nucleic acid-based therapeutics; however, effective patient-friendly delivery remains a major obstacle to clinical utility. A variety of limited and inefficient methods of delivering nucleic acids to keratinocytes have been demonstrated; further advances will require well-characterized reagents, rapid noninvasive assays of delivery, and well-developed skin model systems. Using intravital fluorescence and bioluminescence imaging and a standard set of reporter plasmids we demonstrate transfection of cells in mouse and human xenograft skin using intradermal injection and two microneedle array delivery systems. Reporter gene expression could be detected in individual keratinocytes, in real-time, in both mouse skin as well as human skin xenografts. These studies revealed that non-invasive intravital imaging can be used as a guide for developing gene delivery tools, establishing a benchmark for comparative testing of nucleic acid skin delivery technologies.

Abstract

Development of imaging agents that can be used broadly for early detection of neoplasia at various tissue sites and at various stages of disease and that also can assess states of minimal residual disease would have tremendous utility in the diagnosis and management of cancer. In a series of articles culminating with a report in this issue of the journal (beginning on page 1536), Uddin and colleagues show their ability to systemically target the enzyme COX-2 with imaging probes that will serve as agents for early detection, risk assessment, prognosis, and intervention outcome measures. These probes will enable the detection and localization of regions of inflammation and a wide variety of premalignant lesions and cancers, with utility in monitoring the effects of cancer prevention and therapy.

Abstract

Induction of heat shock protein (Hsp) expression correlates with cytoprotection, reduced tissue damage, and accelerated healing in animal models. Since Hsps are transcriptionally activated in response to stress, they can act as stress indicators in burn injury or surgical procedures that produce heat and thermal change. A fast in vivo readout for induction of Hsp transcription in tissues would allow for the study of these proteins as therapeutic effect mediators and reporters of thermal stress?damage. We used a transgenic reporter mouse in which a luciferase expression is controlled by the regulatory region of the inducible 70 kilodalton (kDa) Hsp as a rapid readout of cellular responses to laser-mediated thermal stress?injury in mouse skin. We assessed the pulse duration dependence of the Hsp70 expression after irradiation with a CO(2) laser at 10.6 ?m in wavelength over a range of 1000 to 1 ms. Hsp70 induction varied with changes in laser pulse durations and radiant exposures, which defined the ranges at which thermal activation of Hsp70 can be used to protect cells from subsequent stress, and reveals the window of thermal stress that tissues can endure.

Abstract

Tuberculosis is a leading cause of death worldwide. Resistance of Mycobacterium to antibiotics can make treatments less effective in some cases. We tested selected oligopeptoids--previously reported as mimics of natural host defense peptides--for activity against Mycobacterium tuberculosis and assessed their cytotoxicity. A tetrameric, alkylated, cationic peptoid (1-C13(4mer)) was most potent against M. tuberculosis and least cytotoxic, whereas an unalkylated analogue, peptoid 1(4mer), was inactive. Peptoid 1-C13(4mer) thus merits further study as a potential antituberculosis drug.

Abstract

The cytoprotective response to thermal injury is characterized by transcriptional activation of "heat shock proteins" (hsp) and proinflammatory proteins. Expression of these proteins may predict cellular survival. Microarray analyses were performed to identify spatially distinct gene expression patterns responding to thermal injury. Laser injury zones were identified by expression of a transgene reporter comprised of the 70 kD hsp gene and the firefly luciferase coding sequence. Zones included the laser spot, the surrounding region where hsp70-luc expression was increased, and a region adjacent to the surrounding region. A total of 145 genes were up-regulated in the laser irradiated region, while 69 were up-regulated in the adjacent region. At 7 hours the chemokine Cxcl3 was the highest expressed gene in the laser spot (24 fold) and adjacent region (32 fold). Chemokines were the most common up-regulated genes identified. Microarray gene expression was successfully validated using qRT- polymerase chain reaction for selected genes of interest. The early response genes are likely involved in cytoprotection and initiation of the healing response. Their regulatory elements will benefit creating the next generation reporter mice and controlling expression of therapeutic proteins. The identified genes serve as drug development targets that may prevent acute tissue damage and accelerate healing.

Abstract

Although RNA interference offers therapeutic potential for treating skin disorders, delivery hurdles have hampered clinical translation. We have recently demonstrated that high pressure, resulting from intradermal injection of large liquid volumes, facilitated nucleic acid uptake by keratinocytes in mouse skin. Furthermore, similar intradermal injections of small interfering RNA (siRNA; TD101) into pachyonychia congenita (PC) patient foot lesions resulted in improvement. Unfortunately, the intense pain associated with hypodermic needle administration to PC lesions precludes this as a viable delivery option for this disorder. To investigate siRNA uptake by keratinocytes, an organotypic epidermal model, in which pre-existing endogenous gene or reporter gene expression can be readily monitored, was used to evaluate the effectiveness of "self-delivery" siRNA (i.e., siRNA chemically modified to enhance cellular uptake). In this model system, self-delivery siRNA treatment resulted in reduction of pre-existing fluorescent reporter gene expression under conditions in which unmodified controls had little or no effect. Additionally, treatment of PC epidermal equivalents with self-delivery "TD101" siRNA resulted in marked reduction of mutant keratin 6a mRNA with little or no effect on wild-type expression. These results indicate that chemical modification of siRNA may overcome certain limitations to transdermal delivery (specifically keratinocyte uptake) and may have clinical utility for inhibition of gene expression in the skin.

Abstract

RNA interference (RNAi) is an evolutionarily conserved mechanism that results in specific gene inhibition at the mRNA level. The discovery that short interfering RNAs (siRNAs) are selective, potent, and can largely avoid immune surveillance has resulted in keen interest to develop these inhibitors as therapeutics. A single nucleotide-specific siRNA (K6a_513a.12, also known as TD101) was recently evaluated in a phase 1b clinical trial for the rare skin disorder, pachyonychia congenita (PC). To develop a clinical trial molecular end point for this type of trial, methods were developed to: (1) isolate total RNA containing amplifiable mRNA from human skin and callus material; (2) quantitatively distinguish the single-nucleotide mutant mRNA from wild-type K6a mRNA in both patient-derived keratinocytes and patient callus; and (3) demonstrate that repeated siRNA treatment results in sustained inhibition of mutant K6a mRNA in patient-derived keratinocyte cultures. These methods allow noninvasive sampling and monitoring of gene expression from patient-collected shavings and may be useful in evaluating the effectiveness of RNAi-based therapeutics, including inhibitors that specifically target single-nucleotide mutations.

Abstract

Advancing molecular therapies for the treatment of skin diseases will require the development of new tools that can reveal spatiotemporal changes in the microanatomy of the skin and associate these changes with the presence of the therapeutic agent. For this purpose, we evaluated a handheld dual-axis confocal (DAC) microscope that is capable of in vivo fluorescence imaging of skin, using both mouse models and human skin. Individual keratinocytes in the epidermis were observed in three-dimensional image stacks after topical administration of near-infrared (NIR) dyes as contrast agents. This suggested that the DAC microscope may have utility in assessing the clinical effects of a small interfering RNA (siRNA)-based therapeutic (TD101) that targets the causative mutation in pachyonychia congenita (PC) patients. The data indicated that (1) formulated indocyanine green (ICG) readily penetrated hyperkeratotic PC skin and normal callused regions compared with nonaffected areas, and (2) TD101-treated PC skin revealed changes in tissue morphology, consistent with reversion to nonaffected skin compared with vehicle-treated skin. In addition, siRNA was conjugated to NIR dye and shown to penetrate through the stratum corneum barrier when topically applied to mouse skin. These results suggest that in vivo confocal microscopy may provide an informative clinical end point to evaluate the efficacy of experimental molecular therapeutics.

Abstract

Molecular imaging is the visualization, characterization, and measurement of biologic processes at the molecular and cellular levels in humans and other living systems (1). It comprises an emerging set of technologies that builds on advances in imaging procedures (e.g., PET, SPECT, MRI, ultrasound, optical, and photoacoustic), improved understanding of biology, and the development of molecularly targeted agents. These continuously expanding sets of imaging methods are often used in combination, and advances in data acquisition and analyses facilitate a more complete understanding of biology. Molecular imaging aims to improve our understanding of mammalian biology and lead to advances in patient care by providing targeted therapies that will enable personalized medicine and the imaging tools to assess outcome. Implementation of these new technologies in clinical care has many educational, technical, and regulatory challenges that must be overcome before molecular imaging reaches its full potential. The impact of molecular imaging has been significant in several disciplines, because it represents a paradigm shift in how scientists and clinicians can observe biology in real time and in a relatively noninvasive manner to enable the power of repeated measures in living organisms.

Abstract

Subthreshold retinal phototherapy demonstrated clinical efficacy for the treatment of diabetic macular edema without visible signs of retinal damage. To assess the range of cellular responses to sublethal hyperthermia, expression of the gene encoding a 70 kDa heat shock protein (HSP70) was evaluated after laser irradiation using a transgenic reporter mouse.One hundred millisecond, 532 nm laser exposures with 400 ?m beam diameter were applied to the retina surrounding the optic nerve in 32 mice. Transcription from the HSP70 promoter was assessed relative to the control eye using a bioluminescence assay at 7 hours after laser application. The retinal pigmented epithelium (RPE) viability threshold was determined with a fluorescence assay. A computational model was developed to estimate temperature and the extent of cell damage.A significant increase in HSP70 transcription was found at exposures over 20 mW, half the threshold power for RPE cell death. Computational modeling estimated peak temperature T = 49°C at HSP70 expression threshold. At RPE viability threshold, T = 57°C. Similar temperatures and damage indices were calculated for clinical subvisible retinal treatment parameters.Beneficial effects of laser therapy have been previously shown to extend beyond those resulting from destruction of tissue. One hundred millisecond laser exposures at approximately half the threshold power of RPE damage induced transcription of HSP70, an indication of cellular response to sublethal thermal stress. A computational model of retinal hyperthermia can guide further optimization of laser parameters for nondamaging phototherapy.

Abstract

Recent advances in optical imaging have led to the development of miniature microscopes that can be brought to the patient for visualizing tissue structures in vivo. These devices have the potential to revolutionize health care by replacing tissue biopsy with in vivo pathology. One of the primary limitations of these microscopes, however, is that the constrained field of view can make image interpretation and navigation difficult. In this paper, we show that image mosaicing can be a powerful tool for widening the field of view and creating image maps of microanatomical structures. First, we present an efficient algorithm for pairwise image mosaicing that can be implemented in real time. Then, we address two of the main challenges associated with image mosaicing in medical applications: cumulative image registration errors and scene deformation. To deal with cumulative errors, we present a global alignment algorithm that draws upon techniques commonly used in probabilistic robotics. To accommodate scene deformation, we present a local alignment algorithm that incorporates deformable surface models into the mosaicing framework. These algorithms are demonstrated on image sequences acquired in vivo with various imaging devices including a hand-held dual-axes confocal microscope, a miniature two-photon microscope, and a commercially available confocal microendoscope.

Abstract

Advances in optical designs are enabling the development of miniature microscopes that can examine tissue in situ for early anatomic and molecular indicators of disease, in real time, and at cellular resolution. These new devices will lead to major changes in how diseases are detected and managed, driving a shift from today's diagnostic paradigm of biopsy followed by histopathology and recommended therapy, to non-invasive point-of-care diagnosis with possible same-session definitive treatment. This shift may have major implications for the training requirements of future physicians to enable them to interpret real-time in vivo microscopic data, and will also shape the emerging fields of telepathology and telemedicine. Implementation of new technologies into clinical practice is a complex process that requires bridging gaps between clinicians, engineers and scientists. This article provides a forward-looking discussion of these issues, with a focus on malignant and pre-malignant lesions, by first highlighting some of the clinical areas where point-of-care in vivo microscopy could address unmet needs, and then by reviewing the technological challenges that are being addressed, or need to be addressed, for in vivo microscopy to become a standard clinical tool.

Abstract

Adoptive immunotherapy is evolving to assume an increasing role in treating cancer. Most imaging studies in adoptive immunotherapy to date have focused primarily on locating tumor-specific T cells rather than understanding their effector functions. In this study, we report the development of a noninvasive imaging strategy to monitor T-cell activation in living subjects by linking a reporter gene to the Granzyme B promoter (pGB), whose transcriptional activity is known to increase during T-cell activation. Because pGB is relatively weak and does not lead to sufficient reporter gene expression for noninvasive imaging, we specifically employed 2 signal amplification strategies, namely the Two Step Transcription Amplification (TSTA) strategy and the cytomegalovirus enhancer (CMVe) strategy, to maximize firefly luciferase reporter gene expression. Although both amplification strategies were capable of increasing pGB activity in activated primary murine splenocytes, only the level of bioluminescence activity achieved with the CMVe strategy was adequate for noninvasive imaging in mice. Using T cells transduced with a reporter vector containing the hybrid pGB-CMVe promoter, we were able to optically image T-cell effector function longitudinally in response to tumor antigens in living mice. This methodology has the potential to accelerate the study of adoptive immunotherapy in preclinical cancer models.

Abstract

Current treatments of high-grade lymphoma often have curative potential, but unfortunately many patients relapse and develop therapeutic resistance. Thus, there remains a need for novel therapeutics that can target the residual cancer cells whose phenotypes are distinct from the bulk tumor and that are capable of reforming tumors from very few cells. Oncolytic viruses offer an approach to destroy tumors by multiple mechanisms, but they cannot effectively reach residual disease or micrometastases, especially within the lymphatic system. To address these limitations, we have generated immune cells infected with oncolytic viruses as a therapeutic strategy that can combine effective cellular delivery with synergistic tumor killing. In this study, we tested this approach against minimal disease states of lymphomas characterized by the persistence of cancer cells that display stem cell-like properties and resistance to conventional therapies. We found that the immune cells were capable of trafficking to and targeting residual cancer cells. The combination biotherapy used prevented relapse by creating a long-term, disease-free state, with acquired immunity to the tumor functioning as an essential mediator of this effect. Immune components necessary for this acquired immunity were identified. We further demonstrated that the dual biotherapy could be applied before or after conventional therapy. Our approach offers a potentially powerful new way to clear residual cancer cells, showing how restoring immune surveillance is critical for maintenance of a disease-free state.

Abstract

Previously, we have shown that a 43°C pretreatment can provide thermotolerance to a following, more severe, thermal stress at 45°C. Using cells that lack the Hsp70 gene, we have also shown that there is still some thermotolerance in the absence of HSP70 protein. The purpose of this study was to determine which genes play a role in thermotolerance by measuring viability and proliferation of the cells at 2 days after heating. Specifically, we wanted to understand which pathways may be responsible for protecting cells in the absence of HSP70.Murine embryonic fibroblast cells with and without Hsp70 (MEF(+/+) and MEF(-/-), respectively) were exposed to a mild heat shock of 43°C for 30?minutes in a constant temperature water bath. After 3?hours of recovery, RNA was harvested from three heated samples alongside three untreated controls using a MicroRNeasy kit with DNAse treatment. RNA quality was verified by an Agilent Bioanalyzer. The RNA was then converted to cDNA and hybridized to Affymetrix gene expression DNA microarrays. The genes that showed a twofold change (up or down) relative to unheated controls were filtered by t-test for significance at a threshold of P?0.05 using Genespring software. Data were verified by qRT-PCR. Genes were then categorized based upon their ontology.While many genes were similarly upregulated, the main difference between cell types was an increase in transcription factors and nucleic acid binding proteins. Several genes known to be involved in the heat response were upregulated more than twofold (Hsp70, Hsp40, Hsp110, Hsp25, Atf3), however, another well studied heat responsive gene Hsp90 only increased by 1.5-fold under these conditions despite its role in thermotolerance.The data herein presents genetic pathways which are candidates for further study of pretreatment protocols in laser irradiation.

Abstract

To examine the role of breast cancer stem cells (BCSCs) in metastasis, we generated human-in-mouse breast cancer orthotopic models using patient tumor specimens, labeled with optical reporter fusion genes. These models recapitulate human cancer features not captured with previous models, including spontaneous metastasis in particular, and provide a useful platform for studies of breast tumor initiation and progression. With noninvasive imaging approaches, as few as 10 cells of stably labeled BCSCs could be tracked in vivo, enabling studies of early tumor growth and spontaneous metastasis. These advances in BCSC imaging revealed that CD44(+) cells from both primary tumors and lung metastases are highly enriched for tumor-initiating cells. Our metastatic cancer models, combined with noninvasive imaging techniques, constitute an integrated approach that could be applied to dissect the molecular mechanisms underlying the dissemination of metastatic CSCs (MCSCs) and to explore therapeutic strategies targeting MCSCs in general or to evaluate individual patient tumor cells and predict response to therapy.

Abstract

Nucleic acid-based therapies hold great promise for treatment of skin disorders if delivery challenges can be overcome. To investigate one mechanism of nucleic acid delivery to keratinocytes, a fixed mass of expression plasmid was intradermally injected into mouse footpads in different volumes, and reporter expression was monitored by intravital imaging or skin sectioning. Reporter gene expression increased with higher delivery volumes, suggesting that pressure drives nucleic acid uptake into cells after intradermal injections similar to previously published studies for muscle and liver. For spatiotemporal analysis of reporter gene expression, a dual-axis confocal (DAC) fluorescence microscope was used for intravital imaging following intradermal injections. Individual keratinocytes expressing hMGFP were readily visualized in vivo and initially appeared to preferentially express in the stratum granulosum and subsequently migrate to the stratum corneum over time. Fluorescence microscopy of frozen skin sections confirmed the patterns observed by intravital imaging. Intravital imaging with the DAC microscope is a noninvasive method for probing spatiotemporal control of gene expression and should facilitate development and testing of new nucleic acid delivery technologies.

Abstract

A key challenge in developing RNAi-based therapeutics is efficient delivery of functional short interfering RNA (siRNA) to target cells. To address this need, we have used a supercritical CO(2) process to incorporate siRNA in biodegradable polymer nanoparticles (NPs) for in vivo sustained release. By this means we have obtained complete encapsulation of the siRNA with minimal initial burst effect from the surface of the NPs. The slow release of a fluorescently labeled siRNA mimic (siGLO Red) was observed for up to 80 days in vivo after intradermal injection into mouse footpads. In vivo gene silencing experiments were also performed, showing reduction of GFP signal in the epidermis of a reporter transgenic mouse model, which demonstrates that the siRNA retained activity following release from the polymer NPs.

Abstract

Despite rapid progress in the development of potent and selective small interfering RNA (siRNA) agents for skin disorders, translation to the clinic has been hampered by the lack of effective, patient-friendly delivery technologies. The stratum corneum poses a formidable barrier to efficient delivery of large and/or charged macromolecules including siRNAs. Intradermal siRNA injection results in effective knockdown of targeted gene expression but is painful and the effects are localized to the injection site. The use of microneedle arrays represents a less painful delivery method and may have utility for the delivery of nucleic acids, including siRNAs. For this purpose, we developed a loadable, dissolvable protrusion array device (PAD) that allows skin barrier penetration. The PAD tips dissolve upon insertion, forming a gel-like plug that releases functional cargo. PAD-mediated delivery of siRNA (modified for enhanced stability and cellular uptake) resulted in effective silencing of reporter gene expression in a transgenic reporter mouse model. PAD delivery of luciferase reporter plasmids resulted in expression in cells of the ear, back, and footpad skin as assayed by intravital bioluminescence imaging. These results support the use of PADs for delivery of functional nucleic acids to cells in the skin with an efficiency that may support clinical translation.

Abstract

A major limitation to the use of immunotherapy in the treatment of cancer has been the localized immune suppressive environment within the tumor. Although there is evidence that tumor-selective (oncolytic) viruses may help to overcome this immune suppression, a primary limitation to their use has been limited systemic delivery potential, especially in the face of antiviral immunity. We recently demonstrated that tumor-trafficking immune cells can efficiently deliver oncolytic viral therapies to their tumor targets. These cells act as both a therapeutic agent and also a carrier vehicle for the oncolytic virus. Here, we demonstrate that such delivery is also possible in the face of pre-existing antiviral immunity, so overcoming the limited systemic delivery of naked, cell-free virus. It was also found that treatment of previously immunized mice or repeat treatments leading to immunization resulted in a switch from a primarily oncolytic to an immunotherapeutic mechanism of action. Furthermore, repeat cycles of treatment with combination immune cell-viral therapy resulted in increased tumor infiltration of effector T-cells and a general reduction in the levels of known immune suppressive lymphocyte populations. This therefore represents a novel and effective means to overcome localized immune suppression within the tumor microenvironment.

Abstract

Transcriptional targeting for cardiac gene therapy is limited by the relatively weak activity of most cardiac-specific promoters. We have developed a bidirectional plasmid vector, which uses a two-step transcriptional amplification (TSTA) strategy to enhance the expression of two optical reporter genes, firefly luciferase (fluc) and Renilla luciferase (hrluc), driven by the cardiac troponin T (cTnT) promoter. The vector was characterized in vitro and in living mice using luminometry and bioluminescence imaging to assess its ability to mediate strong, correlated reporter gene expression in a cardiac cell line and the myocardium, while minimizing expression in non-cardiac cell lines and the liver. In vitro, the TSTA system significantly enhanced cTnT-mediated reporter gene expression with moderate preservation of cardiac specificity. After intramyocardial and hydrodynamic tail vein delivery of an hrluc-enhanced variant of the vector, long-term fluc expression was observed in the heart, but not in the liver. In both the cardiac cell line and the myocardium, fluc expression correlated well with hrluc expression. These results show the vector's ability to effectively amplify and couple transgene expression in a cardiac-specific manner. Further replacement of either reporter gene with a therapeutic gene should allow non-invasive imaging of targeted gene therapy in living subjects.

Abstract

Many pharmaceuticals are formulated as powders to aid drug delivery. A major problem is how to produce powders having high purity, controlled morphology, and retained bioactivity. We demonstrate the use of supercritical carbon dioxide as an antisolvent for meeting this need for two model drug systems, quercetin, a sparingly soluble antioxidant, and short interfering RNA (siRNA), which can silence genes. In both cases we achieve retention of bioactivity as well as a narrow particle size distribution in which the particles are free of impurities.

Abstract

Fourier transform infrared spectroscopy (FTIR) reveals biochemical 'fingerprints' and has found disease patterns in excised human tissues. Fiber-optic probes have been developed for FTIR in living systems, allowing for cancer detection. There are challenges to making in vivo FTIR a reality, which are being addressed through hardware advances, determining key wavelengths and tissue preparation. Fiber-optic evanescent wave spectroscopy (FEWS)-FTIR with endoscope-compatible fiber-optic silver halide probes is feasible, and could prove useful for distinguishing premalignant and malignant tissues from biopsies or within patients. Developments of smaller silver halide probes as well as in vivo tissue drying methods will move this approach closer to the clinic where it can be used for early cancer detection, disease characterization and guided biopsies.

Abstract

Transgenic reporter mice and advances in imaging instrumentation are enabling real-time visualization of cellular mechanisms in living subjects and accelerating the development of novel therapies. Innovative confocal microscope designs are improving their utility for microscopic imaging of fluorescent reporters in living animals. We develop dual-axis confocal (DAC) microscopes for such in vivo studies and create mouse models where fluorescent proteins are expressed in the skin for the purpose of advancing skin therapeutics and transdermal delivery tools. Three-dimensional image volumes, through the different skin compartments of the epidermis and dermis, can be acquired in several seconds with the DAC microscope in living mice, and are comparable to histologic analyses of reporter protein expression patterns in skin sections. Intravital imaging with the DAC microscope further enables visualization of green fluorescent protein (GFP) reporter gene expression in the skin over time, and quantification of transdermal delivery of small interfering RNA (siRNA) and therapeutic efficacy. Visualization of transdermal delivery of nucleic acids will play an important role in the development of innovative strategies for treating skin pathologies.

Abstract

Bacterial targeting of tumours is an important anti-cancer strategy. We previously showed that strain SL7838 of Salmonella typhimurium targets and kills cancer cells. Whether NO generation by the bacteria has a role in SL7838 lethality to cancer cells is explored. This bacterium has the mechanism for generating NO, but also for decomposing it.Mechanism underlying Salmonella typhimurium tumour therapy was investigated through in vitro and in vivo studies. NO measurements were conducted either by chemical assays (in vitro) or using Biosensors (in vivo). Cancer cells cytotoxic assay were done by using MTS. Bacterial cell survival and tumour burden were determined using molecular imaging techniques.SL7838 generated nitric oxide (NO) in anaerobic cell suspensions, inside infected cancer cells in vitro and in implanted 4T1 tumours in live mice, the last, as measured using microsensors. Thus, under these conditions, the NO generating pathway is more active than the decomposition pathway. The latter was eliminated, in strain SL7842, by the deletion of hmp- and norV genes, making SL7842 more proficient at generating NO than SL7838. SL7842 killed cancer cells more effectively than SL7838 in vitro, and this was dependent on nitrate availability. This strain was also ca. 100% more effective in treating implanted 4T1 mouse tumours than SL7838.NO generation capability is important in the killing of cancer cells by Salmonella strains.

Abstract

A fluorescence confocal microscope incorporating a 1.8-mm-diam gradient-index relay lens is developed for in vivo histological guidance during resection of brain tumors. The microscope utilizes a dual-axis confocal architecture to efficiently reject out-of-focus light for high-contrast optical sectioning. A biaxial microelectromechanical system (MEMS) scanning mirror is actuated at resonance along each axis to achieve a large field of view with low-voltage waveforms. The unstable Lissajous scan, which results from actuating the orthogonal axes of the MEMS mirror at highly disparate resonance frequencies, is optimized to fully sample 500x500 pixels at two frames per second. Optically sectioned fluorescence images of brain tissues are obtained in living mice to demonstrate the utility of this microscope for image-guided resections.

Abstract

Bioluminescence imaging (BLI) exploits the light-emitting properties of luciferase enzymes for monitoring cells and biomolecular processes in living subjects. Luciferases can be incorporated into a variety of non-luminescent hosts and used to track cells, visualize gene expression, and analyze collections of biomolecules. This article highlights recent applications of BLI to studies of mammalian biology, along with the development of novel bioluminescent probes to 'see' cells and molecules in action. Collectively, these efforts are expanding our understanding of living systems and shedding light on the molecular underpinnings of disease.

Abstract

We used mast cell-engrafted genetically mast cell-deficient C57BL/6-Kit(W-sh/W-sh) mice to investigate the roles of mast cells and mast cell-derived tumor necrosis factor in two models of severe bacterial infection. In these mice, we confirmed findings derived from studies of mast cell-deficient WBB6F(1)-Kit(W/W-v) mice indicating that mast cells can promote survival in cecal ligation and puncture (CLP) of moderate severity. However, we found that the beneficial role of mast cells in this setting can occur independently of mast cell-derived tumor necrosis factor. By contrast, using mast cell-engrafted C57BL/6-Kit(W-sh/W-sh) mice, we found that mast cell-derived tumor necrosis factor can increase mortality during severe CLP and can also enhance bacterial growth and hasten death after intraperitoneal inoculation of Salmonella typhimurium. In WBB6F(1)-Kit(W-sh/W-sh) mice, mast cells enhanced survival during moderately severe CLP but did not significantly change the survival observed in severe CLP. Our findings in three types of genetically mast cell-deficient mice thus support the hypothesis that, depending on the circumstances (including mouse strain background, the nature of the mutation resulting in a mast cell deficiency, and type and severity of infection), mast cells can have either no detectable effect or opposite effects on survival during bacterial infections, eg, promoting survival during moderately severe CLP associated with low mortality but, in C57BL/6-Kit(W-sh/W-sh) mice, increasing mortality during severe CLP or infection with S. typhimurium.

Abstract

Despite ongoing clinical trials, the optimal time for delivery of bone marrow mononuclear cells (BMCs) after myocardial infarction is unclear. We compared the viability and effects of transplanted BMCs on cardiac function in the acute and subacute inflammatory phases of myocardial infarction.The time course of acute inflammatory cell infiltration was quantified by FACS analysis of enzymatically digested hearts of FVB mice (n=12) after left anterior descending artery ligation. Mac-1(+)Gr-1(high) neutrophil infiltration peaked at day 4. BMCs were harvested from transgenic FVB mice expressing firefly luciferase (Fluc) and green fluorescent protein (GFP). Afterward, 2.5x10(6) BMCs were injected into the left ventricle of wild-type FVB mice either immediately (acute BMC) or 7 days (subacute BMC) after myocardial infarction, or after a sham procedure (n=8 per group). In vivo bioluminescence imaging showed an early signal increase in both BMC groups at day 7, followed by a nonsignificant trend (P=0.203) toward improved BMC survival in the subacute BMC group that persisted until the bioluminescence imaging signal reached<0.01) and 6 weeks (both BMC groups versus saline; P<0.05) but no significant differences between the 2 BMC groups. FACS analysis of BMC-injected hearts at day 7 revealed that GFP(+) BMCs expressed hematopoietic (CD45, Mac-1, Gr-1), minimal progenitor (Sca-1, c-kit), and no endothelial (CD133, Flk-1) or cardiac (Trop-T) cell markers.Timing of BMC delivery has minimal effects on intramyocardial retention and preservation of cardiac function. In general, there is poor long-term engraftment and BMCs tend to adopt inflammatory cell phenotypes.

Abstract

Accurate diagnosis and prognosis is essential for cancer management but is subject to sampling and inter-observer error. In a recent study, Baker et al. compared Fourier transform infrared (FTIR) microspectroscopy with histological pathology to evaluate prostate tissue for disease severity. The authors found that biochemical changes associated with prostate cancer could be discriminated by FTIR to classify confined and locally invasive prostate cancers. These findings could enable the development of improved diagnostic and prognostic methods for the detection and treatment of prostate cancers.

Abstract

Small interfering RNAs (siRNAs) can be designed to specifically and potently target and silence a mutant allele, with little or no effect on the corresponding wild-type allele expression, presenting an opportunity for therapeutic intervention. Although several siRNAs have entered clinical trials, the development of siRNA therapeutics as a new drug class will require the development of improved delivery technologies. In this study, a reporter mouse model (transgenic click beetle luciferase/humanized monster green fluorescent protein) was developed to enable the study of siRNA delivery to skin; in this transgenic mouse, green fluorescent protein reporter gene expression is confined to the epidermis. Intradermal injection of siRNAs targeting the reporter gene resulted in marked reduction of green fluorescent protein expression in the localized treatment areas as measured by histology, real-time quantitative polymerase chain reaction and intravital imaging using a dual-axes confocal fluorescence microscope. These results indicate that this transgenic mouse skin model, coupled with in vivo imaging, will be useful for development of efficient and 'patient-friendly' siRNA delivery techniques and should facilitate the translation of siRNA-based therapeutics to the clinic for treatment of skin disorders.

Abstract

Fourier transform infrared (FTIR) spectroscopy is sensitive to the molecular composition of tissue and has the potential to identify premalignant tissue (dysplasia) as an adjunct to endoscopy. We demonstrate collection of mid-infrared absorption spectra with a silver halide (AgCl(0.4)Br(0.6)) optical fiber and use spectral preprocessing to identify optimal subranges that classify colonic mucosa as normal, hyperplasia, or dysplasia. We collected spectra (n=83) in the 950 to 1800 cm(-1) regime on biopsy specimens obtained from human subjects (n=37). Subtle differences in the magnitude of the absorbance peaks at specific wave numbers were observed. The best double binary algorithm for distinguishing normal-versus-dysplasia and hyperplasia-versus-dysplasia was determined from an exhaustive search of spectral intervals and preprocessing techniques. Partial least squares discriminant analysis was used to classify the spectra using a leave-one-subject-out cross-validation strategy. The results were compared with histology reviewed independently by two gastrointestinal pathologists. The optimal thresholds identified resulted in an overall sensitivity, specificity, accuracy, and positive predictive value of 96%, 92%, 93%, and 82%, respectively. These results indicated that mid-infrared absorption spectra collected remotely with an optical fiber can be used to identify colonic dysplasia with high accuracy, suggesting that continued development of this technique for the early detection of cancer is promising.

Abstract

Liposomal formulations of drugs have been shown to enhance drug efficacy by prolonging circulation time, increasing local concentration and reducing off-target effects. Controlled release from these formulations would increase their utility, and hyperthermia has been explored as a stimulus for targeted delivery of encapsulated drugs. Use of lasers as a thermal source could provide improved control over the release of the drug from the liposomes with minimal collateral tissue damage. Appropriate methods for assessing local release after systemic delivery would aid in testing and development of better formulations. We use in vivo bioluminescence imaging to investigate the spatiotemporal distribution of luciferin, used as a model small molecule, and demonstrate laser-induced release from liposomes in animal models after systemic delivery. These liposomes were tested for luciferin release between 37 and 45 degrees C in PBS and serum using bioluminescence measurements. In vivo studies were performed on transgenic reporter mice that express luciferase constitutively throughout the body, thus providing a noninvasive readout for controlled release following systemic delivery. An Nd:YLF laser was used (527 nm) to heat tissues and induce rupture of the intravenously delivered liposomes in target tissues. These data demonstrate laser-mediated control of small molecule delivery using thermally sensitive liposomal formulations.

Abstract

Because of their potent immunoregulatory capacity, dendritic cells (DCs) have been exploited as therapeutic tools to boost immune responses against tumors or pathogens, or dampen autoimmune or allergic responses. Murine bone marrow-derived DCs (BM-DCs) are the closest known equivalent of the blood monocyte-derived DCs that have been used for human therapy. Current imaging methods have proven unable to properly address the migration of injected DCs to small and deep tissues in mice and humans. This study presents the first extensive analysis of BM-DC homing to lymph nodes (and other selected tissues) after intravenous and intraperitoneal inoculation. After intravenous delivery, DCs accumulated in the spleen, and preferentially in the pancreatic and lung-draining lymph nodes. In contrast, DCs injected intraperitoneally were found predominantly in peritoneal lymph nodes (pancreatic in particular), and in omentum-associated lymphoid tissue. This uneven distribution of BM-DCs, independent of the mouse strain and also observed within pancreatic lymph nodes, resulted in the uneven induction of immune response in different lymphoid tissues. These data have important implications for the design of systemic cellular therapy with DCs, and in particular underlie a previously unsuspected potential for specific treatment of diseases such as autoimmune diabetes and pancreatic cancer.

Abstract

The delicate balance between protective immunity and inflammatory disease is challenged during sepsis, a pathologic state characterized by aspects of both a hyperactive immune response and immunosuppression. The events driven by systemic infection by bacterial pathogens on the T cell signaling network that likely control these responses have not been illustrated in great detail. We characterized how intracellular signaling within the immune compartment is reprogrammed at the single cell level when the host is challenged with a high level of pathogen. To accomplish this, we applied flow cytometry to measure the phosphorylation potential of key signal transduction proteins during acute bacterial challenge. We modeled the onset of sepsis by i.v. administration of avirulent strains of Listeria monocytogenes and Escherichia coli to mice. Within 6 h of bacterial challenge, T cells were globally restricted in their ability to respond to specific cytokine stimulations as determined by assessing the extent of STAT protein phosphorylation. Mechanisms by which this negative feedback response occurred included SOCS1 and SOCS3 gene up-regulation and IL-6-induced endocystosis of the IL-6 receptor. Additionally, macrophages were partially tolerized in their ability to respond to TLR agonists. Thus, in contrast to the view that there is a wholesale immune activation during sepsis, one immediate host response to blood-borne bacteria was induction of a refractory period during which leukocyte activation by specific stimulations was attenuated.

Abstract

The burgeoning fields of in vivo three-dimensional (3D) microscopy and endomicroscopy, as well as ex vivo tissue cytometry have introduced new challenges for tissue preparation and staining with exogenous molecular contrast agents. These challenges include effective delivery of the agents, and once delivered, distinguishing between bound verses unbound molecular probes. If applied topically, there are additional issues with rinsing off unbound probe, which can be nonuniform and inefficient in thick tissues, thus leading to ambiguous contrast and a large nonspecific background that may obscure molecule-specific staining. Therefore, we have developed a ratiometric 3D microscopy scheme that not only reduces the effects of nonspecific sources of contrast, but also enables quantification of the relative binding affinity of imaging probes to their biomarker targets. Here we demonstrate this ratiometric approach by simultaneously imaging a HER2/neu (erbB2)-targeted monoclonal antibody labeled with one fluorophore and an isotype-matched negative control antibody labeled with another fluorophore. By taking a pixel-by-pixel calibrated ratio between the signals from each fluorescent image channel, accurate quantification of specific versus nonspecific binding affinity is achieved with cultured cells, yielding data that are in agreement with analyses via flow cytometry. We also demonstrate quantitative 3D microscopic imaging of biomarker expression in tissue models and in thick human biopsy samples of normal, HER2-negative, and HER2-positive breast tumors. This strategy enables rapid, quantitative, and unambiguous volumetric microscopy of biomarker expression in thick tissues, including whole biopsies, and will enable real-time optical assessment of disease markers in the living body.

Abstract

Mesenchymal stem cells hold promise for cardiovascular regenerative therapy. Derivation of these cells from the adipose tissue might be easier compared with bone marrow. However, the in vivo fate and function of adipose stromal cells (ASC) in the infarcted heart has never been compared directly to bone marrow-derived mesenchymal cells (MSC).ASC and MSC were isolated from transgenic FVB mice with a beta-actin promoter driving firefly luciferase and green fluorescent protein double fusion reporter gene, and they were characterized using flow cytometry, microscopy, bioluminescence imaging and luminometry. FVB mice (n=8 per group) underwent myocardial infarction followed by intramyocardial injection of 5x10(5) ASC, MSC, fibroblasts (Fibro, positive control), or saline (negative control). Cell survival was measured using bioluminescence imaging for 6 weeks and cardiac function was monitored by echocardiography and pressure-volume analysis. Ventricular morphology was assessed using histology.ASC and MSC were CD34(-), CD45(-), c-Kit(-), CD90(+), Sca-1(+), shared similar morphology and had a population doubling time of approximately 2 days. Cells expressed Fluc reporter genes in a number-dependent fashion as confirmed by luminometry. After cardiac transplantation, both cell types showed drastic donor cell death within 4 to 5 weeks. Furthermore, transplantation of either cell type was not capable of preserving ventricular function and dimensions, as confirmed by pressure-volume-loops and histology.This is the first study comparing the in vivo behavior of both cell types in the infarcted heart. ASC and MSC do not tolerate well in the cardiac environment, resulting in acute donor cell death and a subsequent loss of cardiac function similar to control groups.

Abstract

Adult stem cells are promising therapeutic reagents for skeletal regeneration. We hope to validate by molecular imaging technologies the in vivo life cycle of adipose-derived multipotent cells (ADMCs) in an animal model of skeletal injury. Primary ADMCs were lentivirally transfected with a fusion reporter gene and injected intravenously into mice with bone injury or sham operation. Bioluminescence imaging (BLI), [(18)F]FHBG (9-(fluoro-hydroxy-methyl-butyl-guanine)-micro-PET, [(18)F]Fluoride ion micro-PET and micro-CT were performed to monitor stem cells and their effect. Bioluminescence microscopy and immunohistochemistry were done for histological confirmation. BLI showed ADMC's traffic from the lungs then to the injury site. BLI microscopy and immunohistochemistry confirmed the ADMCs in the bone defect. Micro-CT measurements showed increased bone healing in the cell-injected group compared to the noninjected group at postoperative day 7 (p < 0.05). Systemically administered ADMC's traffic to the site of skeletal injury and facilitate bone healing, as demonstrated by molecular and small animal imaging. Molecular imaging technologies can validate the usage of adult adipose tissue-derived multipotent cells to promote fracture healing. Imaging can in the future help establish therapeutic strategies including dosage and administration route.

Abstract

The inadequacy of white-light cystoscopy to detect flat bladder tumors is well recognized. Great interest exists in developing other imaging technologies to augment or supplant conventional cystoscopy. Fibered confocal microscopy offers the promise of providing in vivo histopathologic information to help distinguish malignant from benign bladder lesions. We report the initial use of this technology to visualize tumors in the human bladder.We performed ex vivo fibered confocal imaging of fresh radical cystectomy specimens using the Mauna Kea Technologies Cellvizio system. The findings were compared with results from standard histopathology.The bladders of four patients were imaged using the fibered confocal microscope. Normal and neoplastic urothelium manifested differences in cellular and vascular density.This study demonstrates the feasibility of using fibered confocal microscopy to detect histologic differences between normal and neoplastic urothelium, and establishes a foundation for the use of fiber-based confocal microscopy in clinical studies.

Abstract

We report the discovery of a new prodrug, 6-chloro-9-nitro-5-oxo-5H-benzo(a)phenoxazine (CNOB). This prodrug is efficiently activated by ChrR6, the highly active prodrug activating bacterial enzyme we have previously developed. The CNOB/ChrR6 therapy was effective in killing several cancer cell lines in vitro. It also efficiently treated tumors in mice with up to 40% complete remission. 9-Amino-6-chloro-5H-benzo(a)phenoxazine-5-one (MCHB) was the only product of CNOB reduction by ChrR6. MCHB binds DNA; at nonlethal concentration, it causes cell accumulation in the S phase, and at lethal dose, it induces cell surface Annexin V and caspase-3 and caspase-9 activities. Further, MCHB colocalizes with mitochondria and disrupts their electrochemical potential. Thus, killing by CNOB involves MCHB, which likely induces apoptosis through the mitochondrial pathway. An attractive feature of the CNOB/ChrR6 regimen is that its toxic product, MCHB, is fluorescent. This feature proved helpful in in vitro studies because simple fluorescence measurements provided information on the kinetics of CNOB activation within the cells, MCHB killing mechanism, its generally efficient bystander effect in cells and cell spheroids, and its biodistribution. The emission wavelength of MCHB also permitted its visualization in live animals, allowing noninvasive qualitative imaging of MCHB in mice and the tumor microenvironment. This feature may simplify exploration of barriers to the penetration of MCHB in tumors and their amelioration.

Abstract

Matrix metalloproteinase-14 (MT1-MMP or MMP-14) is a membrane-associated protease implicated in a variety of tissue remodeling processes and a molecular hallmark of select metastatic cancers. The ability to detect MMP-14 in vivo would be useful in studying its role in pathologic processes and may potentially serve as a guide for the development of targeted molecular therapies. Four MMP-14 specific probes containing a positively charged cell penetrating peptide (CPP) d-arginine octamer (r(8)) linked with a MMP-14 peptide substrate and attenuating sequences with glutamate (8e, 4e) or glutamate-glycine (4eg and 4egg) repeating units were modeled using an AMBER force field method. The probe with 4egg attenuating sequence exhibited the highest CPP/attenuator interaction, predicting minimized cellular uptake until cleaved. The in vitro MMP-14-mediated cleavage studies using the human recombinant MMP-14 catalytic domain revealed an enhanced cleavage rate that directly correlated with the linearity of the embedded peptide substrate sequence. Successful cleavage and uptake of a technetium-99m labeled version of the optimal probe was demonstrated in MMP-14 transfected human breast cancer cells. Two-fold reduction of cellular uptake was found in the presence of a broad spectrum MMP inhibitor. The combination of computational chemistry, parallel synthesis and biochemical screening, therefore, shows promise as a set of tools for developing new radiolabeled probes that are sensitive to protease activity.

Abstract

Murine listeriosis is one of the most comprehensive and well-studied models of infection, and Listeria monocytogenes has provided seminal information regarding bacterial pathogenesis. However, many aspects of the mouse model remain poorly understood, including carrier states and chronic colonization which represent important features of the spectrum of host-pathogen interaction. Bone marrow has recently been shown to harbor L. monocytogenes, which spreads from this location to the central nervous system. Bone could, therefore, be an important chronic reservoir, but this infection is difficult to study because it involves only a few bacteria and the extent of infection cannot be assessed until after the animal is sacrificed. We employed in vivo bioluminescence imaging to localize L. monocytogenes bone infections over time in live mice, revealing that the bacteria grow in discrete foci. These lesions can persist in many locations in the legs of mice and are not accompanied by a histological indication such as granuloma or a neutrophil infiltratate. We demonstrate that highly attenuated hly mutants, which have defective intracellular replication, are capable of prolonged focal infection of the bone marrow for periods of up to several weeks. These results support the recently proposed hypothesis that the bone marrow is a unique niche for L. monocytogenes.

Abstract

The objective of the study was to track the distribution and differentiation of mesenchymal stem cells (MSCs) in tumor-bearing mice. The 4T1 murine breast cancer cells were labeled with renilla luciferase-monomeric red fluorescence protein (rLuc-mRFP) reporter gene. The MSCs labeled with firefly luciferase-enhanced green fluorescence protein (fLuc-eGFP) reporter gene (MSCs-R) were isolated from L2G85 transgenic mice that constitutively express fLuc-eGFP reporter gene. To study the tumor tropism of MSCs, we established both subcutaneous and lung metastasis models. In lung metastasis tumor mice, we injected MSCs-R intravenously either on the same day or 4 days after 4T1 tumor cell injection. In subcutaneous tumor mice, we injected MSCs-R intravenously 7 days after subcutaneous 4T1 tumor inoculation. The tumor growth was monitored by rLuc bioluminescence imaging (BLI). The fate of MSCs-R was monitored by fLuc BLI. The localization of MSCs-R in tumors was examined histologically. The osteogenic and adipogenic differentiation of MSCs-R was investigated by alizarin red S and oil red O staining, respectively. The mechanism of the dissimilar differentiation potential of MSCs-R under different tumor microenvironments was investigated. We found that the 4T1 cells were successfully labeled with rLuc-mRFP. The MSCs-R isolated from L2G85 transgenic mice constitutively express fLuc-eGFP reporter gene. When injected intravenously, MSCs-R survived, proliferated, and differentiated in tumor sites but not elsewhere. The localization of GFP(+) MSCs-R in tumor lesions was confirmed ex vivo. In conclusion, the MSCs-R can selectively localize, survive, and proliferate in both subcutaneous tumor and lung metastasis as evidenced by noninvasive bioluminescence imaging and ex vivo validation. The MSCs-R migrated to lung tumor differentiated into osteoblasts, whereas the MSCs-R targeting subcutaneous tumor differentiated into adipocytes.

Abstract

An effective response to extreme hematopoietic stress requires an extreme elevation in hematopoiesis and preservation of hematopoietic stem cells (HSCs). These diametrically opposed processes are likely to be regulated by genes that mediate cellular adaptation to physiologic stress. Herein, we show that heme oxygenase-1 (HO-1), the inducible isozyme of heme degradation, is a key regulator of these processes. Mice lacking one allele of HO-1 (HO-1(+/-)) showed accelerated hematopoietic recovery from myelotoxic injury, and HO-1(+/-) HSCs repopulated lethally irradiated recipients with more rapid kinetics. However, HO-1(+/-) HSCs were ineffective in radioprotection and serial repopulation of myeloablated recipients. Perturbations in key stem cell regulators were observed in HO-1(+/-) HSCs and hematopoietic progenitors (HPCs), which may explain the disrupted response of HO-1(+/-) HPCs and HPCs to acute stress. Control of stem cell stress response by HO-1 presents opportunities for metabolic manipulation of stem cell-based therapies.

Abstract

RNA interference offers enormous potential to develop therapeutic agents for a variety of diseases. To assess the stability of siRNAs under conditions relevant to clinical use with particular emphasis on topical delivery considerations, a study of three different unmodified siRNAs was performed. The results indicate that neither repeated freeze/thaw cycles, extended incubations (over 1 year at 21 degrees C), nor shorter incubations at high temperatures (up to 95 degrees C) have any effect on siRNA integrity as measured by nondenaturing polyacrylamide gel electrophoresis and functional activity assays. Degradation was also not observed following exposure to hair or skin at 37 degrees C. However, incubation in fetal bovine or human sera at 37 degrees C led to degradation and loss of activity. Therefore, siRNA in the bloodstream is likely inactivated, thereby limiting systemic exposure. Interestingly, partial degradation (observed by gel electrophoresis) did not always correlate with loss of activity, suggesting that partially degraded siRNAs retain full functional activity. To demonstrate the functional activity of unmodified siRNA, EGFP-specific inhibitors were injected into footpads and shown to inhibit preexisting EGFP expression in a transgenic reporter mouse model. Taken together, these data indicate that unmodified siRNAs are viable therapeutic candidates.

Abstract

Thermal pretreatment has been shown to condition tissue to a more severe secondary heat stress. In this research we examined the particular contribution of heat shock protein 70 (HSP70) in thermal preconditioning.For optimization of preshock exposures, a bioluminescent Hsp70-luciferase reporter system in NIH3T3 cells tracked the activation of the Hsp70 gene. Cells in 96-well plates were pretreated in a 43 degrees C water bath for 30 minutes, followed 4 hours later with a severe heat shock at 45 degrees C for 50 minutes. Bioluminescence was measured at 2, 4, 6, 8, and 10 hours after preshock only (PS) and at 4 hours after preshock with heatshock (PS+HS). Viability was assessed 48 hours later with a fluorescent viability dye. Preshock induced thermotolerance was then evaluated in hsp70-containing Murine Embryo Fibroblast (+/+) cells and Hsp70-deficient MEF cells (-/-) through an Arrhenius damage model across varying temperatures (44.5-46 degrees C).A time gap of 4 hours between preconditioning and the thermal insult was shown to be the most effective for thermotolerance with statistical confidence of P<0.05. The benefit of preshocking was largely abrogated in Hsp70-deficient cells. The Arrhenius data showed that preshocking leads to increases in the activation energies, E(a), and increases in frequency factors, A. The frequency factor increase was significantly greater in Hsp70-deficient cells.The data shows that HSP70 contributes significantly to cellular thermotolerance but there are other pathways that provide residual thermotolerance in cells deficient in Hsp70.

Abstract

Conditional control of protein function in vivo offers great potential for deconvoluting the roles of individual proteins in complicated systems. We recently developed a method in which a small protein domain, termed a destabilizing domain, confers instability to fusion protein partners in cultured cells. Instability is reversed when a cell-permeable small molecule binds this domain. Here we describe the use of this system to regulate protein function in living mammals. We show regulation of secreted proteins and their biological activity with conditional secretion of an immunomodulatory cytokine, resulting in tumor burden reduction in mouse models. Additionally, we use this approach to control the function of a specific protein after systemic delivery of the gene that encodes it to a tumor, suggesting uses for enhancing the specificity and efficacy of targeted gene-based therapies. This method represents a new strategy to regulate protein function in living organisms with a high level of control.

Abstract

A comparative analysis of the efficacy of different cell candidates for the treatment of heart disease remains to be described. This study is designed to evaluate the therapeutic efficacy of 4 cell types in a murine model of myocardial infarction.Bone marrow mononuclear cells (MN), mesenchymal stem cells (MSC), skeletal myoblasts (SkMb), and fibroblasts (Fibro) expressing firefly luciferase (Fluc) and green fluorescence protein (GFP) were characterized by flow cytometry, bioluminescence imaging (BLI), and luminometry. Female FVB mice (n=70) underwent LAD ligation and intramyocardially received one cell type (5x10(5)) or PBS. Cell survival was measured by BLI and by TaqMan PCR. Cardiac function was assessed by echocardiography and invasive hemodynamic measurements. Fluc expression correlated with cell number in all groups (r(2)>0.93). In vivo BLI revealed acute donor cell death of MSC, SkMb, and Fibro within 3 weeks after transplantation. By contrast, cardiac signals were still present after 6 weeks in the MN group, as confirmed by TaqMan PCR (P<0.01). Echocardiography showed significant preservation of fractional shortening in the MN group compared to controls (P<0.05). Measurements of left ventricular end-systolic/diastolic volumes revealed that the least amount of ventricular dilatation occurred in the MN group (P<0.05). Histology confirmed the presence of MN, although there was no evidence of transdifferentiation by donor MN into cardiomyocytes.This is the first study to show that compared to MSC, SkMB, and Fibro, MN exhibit a more favorable survival pattern, which translates into a more robust preservation of cardiac function.

Abstract

Laser surgical ablation is achieved by selecting laser parameters that remove confined volumes of target tissue and cause minimal collateral damage. Previous studies have measured the effects of wavelength on ablation, but neglected to measure the cellular impact of ablation on cells outside the lethal zone. In this study, we use optical imaging in addition to conventional assessment techniques to evaluate lethal and sublethal collateral damage after ablative surgery with a free-electron laser (FEL). Heat shock protein (HSP) expression is used as a sensitive quantitative marker of sublethal damage in a transgenic mouse strain, with the hsp70 promoter driving luciferase and green fluorescent protein (GFP) expression (hsp70A1-L2G). To examine the wavelength dependence in the mid-IR, laser surgery is conducted on the hsp70A1-L2G mouse using wavelengths targeting water (OH stretch mode, 2.94 microm), protein (amide-II band, 6.45 microm), and both water and protein (amide-I band, 6.10 microm). For all wavelengths tested, the magnitude of hsp70 expression is dose-dependent and maximal 5 to 12 h after surgery. Tissues treated at 6.45 microm have approximately 4x higher hsp70 expression than 6.10 microm. Histology shows that under comparable fluences, tissue injury at the 2.94-microm wavelength was 2x and 3x deeper than 6.45 and 6.10 microm, respectively. The 6.10-microm wavelength generates the least amount of epidermal hyperplasia. Taken together, this data suggests that the 6.10-microm wavelength is a superior wavelength for laser ablation of skin.

Abstract

Many cancer therapeutic agents elicit resistance that renders them ineffective and often produces cross-resistance to other drugs. One of the most common mechanisms of resistance involves P-glycoprotein (Pgp)-mediated drug efflux. To address this problem, new agents have been sought that are less prone to inducing resistance and less likely to serve as substrates for Pgp efflux. An alternative to this approach is to deliver established agents as molecular transporter conjugates into cells through a mechanism that circumvents Pgp-mediated efflux and allows for release of free drug only after cell entry. Here we report that the widely used chemotherapeutic agent Taxol, ineffective against Taxol-resistant human ovarian cancer cell lines, can be incorporated into a releasable octaarginine conjugate that is effective against the same Taxol-resistant cell lines. It is significant that the ability of the Taxol conjugates to overcome Taxol resistance is observed both in cell culture and in animal models of ovarian cancer. The generality and mechanistic basis for this effect were also explored with coelenterazine, a Pgp substrate. Although coelenterazine itself does not enter cells because of Pgp efflux, its octaarginine conjugate does so readily. This approach shows generality for overcoming the multidrug resistance elicited by small-molecule cancer chemotherapeutics and could improve the prognosis for many patients with cancer and fundamentally alter search strategies for novel therapeutic agents that are effective against resistant disease.

Abstract

The development of transgenic reporter mice and advances in in vivo optical imaging have created unique opportunities to assess and analyze biological responses to thermal therapy directly in living tissues. Reporter mice incorporating the regulatory regions from the genes encoding the 70 kDa heat-shock proteins (Hsp70) and firefly luciferase (luc) as reporter genes can be used to non-invasively reveal gene activation in living tissues in response to thermal stress. High-intensity-focused ultrasound (HIFU) can deliver measured doses of acoustic energy to highly localized regions of tissue at intensities that are sufficient to stimulate Hsp70 expression. We report activation of Hsp70-luc expression using 1 s duration HIFU heating to stimulate gene expression in the skin of the transgenic reporter mouse. Hsp70 expression was tracked for 96 h following the application of 1.5 MHz continuous-wave ultrasound with spatial peak intensities ranging from 53 W cm(-2) up to 352 W cm(-2). The results indicated that peak Hsp70 expression is observed 6-48 h post-heating, with significant activity remaining at 96 h. Exposure durations were simulated using a finite-element model, and the predicted temperatures were found to be consistent with the observed Hsp70 expression patterns. Histological evaluation revealed that the thermal damage starts at the stratum corneum and extends deeper with increasing intensity. These results indicated that short-duration HIFU may be useful for inducing heat-shock expression, and that the period between treatments needs to be greater than 96 h due to the protective properties of Hsp70.

Abstract

We present a handheld dual-axes confocal microscope that is based on a two-dimensional microelectromechanical systems (MEMS) scanner. It performs reflectance and fluorescence imaging at 488 nm wavelength, with three-dimensional imaging capability. The fully packaged microscope has a diameter of 10 mm and acquires images at 4 Hz frame rate with a maximum field of view of 400 microm x 260 microm. The transverse and axial resolutions of the handheld probe are 1.7 microm and 5.8 microm, respectively. Capability to perform real time small animal imaging is demonstrated in vivo in transgenic mice.

Abstract

Miniature endoscopic microscopes, with subcellular imaging capabilities, will enable in vivo detection of molecularly-targeted fluorescent probes for early disease detection. To optimize a dual-axis confocal microscope (DACM) design for this purpose, we use a tabletop instrument to determine the ability of this technology to perform optical sectioning deep within tissue. First, we determine how tissue scattering deteriorates the diffraction-limited transverse and vertical responses in reflectance imaging. Specifically, the vertical response of a DACM to a plane reflector is measured at various depths in a scattering phantom and compared with diffraction theory and Monte Carlo scattering simulations. Similarly, transverse line scans across a knife-edge target are performed at various depths in a scattering phantom. Second, as a practical demonstration of deep-tissue fluorescence microscopy that corroborates the findings from our scattering experiments, 3-D fluorescence images are obtained in thick human gastrointestinal mucosal specimens. Our results demonstrate efficient rejection of scattered light in a DACM, which enables deep optical sectioning in tissue with subcellular resolution that can distinguish between normal and premalignant pathologies.

Abstract

Induction of heat shock protein (Hsp) expression appears to correlate with a cytoprotective effect in cultured cells and with improved healing of damaged tissues in animal models and in humans. This family of proteins can also serve as indicators of thermal stress in cases of burn injury or surgical procedures that produce heat. Thus, a rapid in vivo readout for induction of Hsp transcription would facilitate studies of Hsp genes and their encoded proteins as mediators of therapeutic effects and as reporters of thermal damage to tissues. We created a transgenic reporter mouse where expression of luciferase is controlled by the regulatory region of the inducible 70 kDa Hsp, and assessed activation of Hsp70 transcription in live animals in response to rapid, high temperature stresses using in vivo bioluminescence imaging (BLI). This model can be used to noninvasively reveal levels of Hsp70 transcription in living tissues, and has utility in studies of the predictive and protective effects of Hsp70 expression, and of various stress responses in tissues.

Abstract

Despite significant advances in the development of tumor-selective agents, strategies for effective delivery of these agents across biological barriers to cells within the tumor microenvironment has been limiting. One tactical approach to overcoming biological barriers is to use cells as delivery vehicles, and a variety of different cell types have been investigated with a range of agents. In addition to transporting agents with targeted delivery, cells can also produce their own tumoricidal effect, conceal a payload from an immune response, amplify a selective agent at the target site and facilitate an antitumor immune response. We have reported a therapeutic combination consisting of cytokine induced killer cells and an oncolytic vaccinia virus with many of these features that led to therapeutic synergy in animal models of human cancer. The synergy was due to the interaction of the two agents to enhance the antitumor benefits of each individual component. As both of these agents display broad tumor-targeting potential and possess unique tumor killing mechanisms, together they were able to recognize and destroy a far greater number of malignant cells within the heterogeneous tumor than either agent alone. Effective cancer therapy will require recognition and elimination of the root of the disease, the cancer stem cell, and the combination of CIK cells and oncolytic vaccinia viruses has this potential. To create effective tumor-selective agents the viruses are modified to take advantage of the unique biology of the cancer cell. Similarly, if we are to develop targeted therapies that are sufficiently multifaceted to eliminate cancer cells at all stages of disease, we should integrate the virus into the unique biology of the cell delivery vehicle.

Abstract

Heme oxygenase (HMOX) regulates vascular tone and blood pressure through the production of carbon monoxide (CO), a vasodilator derived from the heme degradation pathway. During pregnancy, the maternal circulation undergoes significant adaptations to accommodate the hemodynamic demands of the developing fetus. Our objective was to investigate the role of HMOX on maternal and fetal hemodynamics during pregnancy in a mouse model. We measured and compared maternal tissue and placental HMOX activity and endogenous CO production, represented by excreted CO and carboxyhemoglobin levels, during pregnancy (Embryonic Days 12.5-15.5) to nonpregnant controls. Micro-ultrasound was used to monitor maternal abdominal aorta diameters as well as blood flow velocities and diameters of fetal umbilical arteries. Tin mesoporphyrin, a potent HMOX inhibitor, was used to inhibit HMOX activity. Changes in maternal vascular tone were monitored by tail cuff blood pressure measurements. Effects of HMOX inhibition on placental structures were assessed by histology. We showed that maternal tissue and placental HMOX activity and CO production were significantly elevated during pregnancy. When HMOX in the placenta was inhibited, maternal and fetal hemodynamics underwent significant changes, with maternal blood pressures increasing. We concluded that increases in maternal tissue and placental HMOX activity contribute to the regulation of peripheral vascular resistance and therefore are important for the maintenance of normal maternal vascular tone and fetal hemodynamic functions during pregnancy.

Abstract

The antitumoral effects of oncolytic viruses have generally been limited by inefficient spread of the viruses within infected tumors and by inefficient systemic delivery, particularly in preimmunized hosts. Tumor-selective poxviruses have biological characteristics that may overcome these limitations. Nevertheless, physical barriers within the tumor microenvironment, including the extracellular matrix, can still limit intratumoral spread, and neutralizing antibodies can impede systemic delivery. To counter these limitations, we sought to take advantage of a naturally occurring poxvirus form known as extracellular enveloped virus (EEV). The EEV is shrouded by a host cell-derived lipid bilayer containing anticomplement proteins and is typically released from infected cells early during the infection cycle. Therefore, the EEV form evolved for rapid systemic spread within the host and for evasion of immune-mediated clearance. We compared the oncolytic potential of low versus high EEV-producing strains of vaccinia. EEV-enhanced vaccinia strains displayed improved spread within tumors after systemic delivery, resulting in significantly improved antitumor effects. The EEV-enhanced strains also displayed a greater ability to spread between injected and noninjected distant tumors through the blood and, importantly, displayed reduced clearance by neutralizing antibody. Safety was unaffected. The incorporation of EEV-enhancing mutations into next generation oncolytic vaccinia strains may improve the potency of these viruses without sacrificing safety.

Abstract

A combination of targeted probes and new imaging technologies provides a powerful set of tools with the potential to improve the early detection of cancer. To develop a probe for detecting colon cancer, we screened phage display peptide libraries against fresh human colonic adenomas for high-affinity ligands with preferential binding to premalignant tissue. We identified a specific heptapeptide sequence, VRPMPLQ, which we synthesized, conjugated with fluorescein and tested in patients undergoing colonoscopy. We imaged topically administered peptide using a fluorescence confocal microendoscope delivered through the instrument channel of a standard colonoscope. In vivo images were acquired at 12 frames per second with 50-microm working distance and 2.5-microm (transverse) and 20-microm (axial) resolution. The fluorescein-conjugated peptide bound more strongly to dysplastic colonocytes than to adjacent normal cells with 81% sensitivity and 82% specificity. This methodology represents a promising diagnostic imaging approach for the early detection of colorectal cancer and potentially of other epithelial malignancies.

Abstract

RNA interference offers a novel approach for developing therapeutics for dominant-negative genetic disorders. The ability to inhibit expression of the mutant allele without affecting wild-type gene expression could be a powerful new treatment option. Targeting the single-nucleotide keratin 6a (K6a) N171K mutation responsible for the rare monogenic skin disorder pachyonychia congenita (PC), we demonstrate that small interfering RNAs (siRNAs) can potently and selectively block expression of mutant K6a. To test whether lead siRNAs could discriminate mutant mRNA in the presence of both wild-type and mutant forms, a dominant-negative PC cell culture model was developed. As predicted for a dominant-negative disease, simultaneous expression of both wild-type and mutant K6a resulted in defective keratin filament formation. Addition of mutant-specific siRNAs allowed normal filament formation, suggesting selective inhibition of mutant K6a. The effectiveness of our siRNA in skin was tested by co-delivering a firefly luciferase/mutant K6a bicistronic reporter construct and mutant-specific siRNAs to mouse footpads. Potent inhibition of the fluorescent reporter was demonstrated using the Xenogen IVIS200 in vivo imaging system. Additionally, wild type-specific siRNAs knocked down the expression of pre-existing endogenous K6a in human keratinocytes. These results suggest that efficient delivery of these "designer siRNAs" may allow effective treatment of numerous genetic disorders including PC.

Abstract

Pachyonychia congenita (PC) is an autosomal-dominant keratin disorder where the most painful, debilitating aspect is plantar keratoderma. PC is caused by mutations in one of four keratin genes; however, most patients carry K6a mutations. Knockout mouse studies suggest that ablation of one of the several K6 genes can be tolerated owing to compensatory expression of the others. Here, we have developed potent RNA interference against K6a as a paradigm for treating a localized dominant skin disorder. Four small interfering RNAs (siRNAs) were designed against unique sequences in the K6a 3'-untranslated region. We demonstrated near-complete ablation of endogenous K6a protein expression in two keratinocyte cell lines, HaCaT and NEB-1, by transient transfection of each of the four K6a siRNAs. The siRNAs were effective at very low, picomolar concentrations. One potent lead K6a inhibitor, which was highly specific for K6a, was tested in a mouse model where reporter gene constructs were injected intradermally into mouse paw and luciferase activity was used as an in vivo readout. Imaging in live mice using the Xenogen IVIS system demonstrated that the K6a-specific siRNA strongly inhibited bicistronic K6a-luciferase gene expression in vivo. These data suggest that siRNAs can specifically and very potently target mutated genes in the skin and support development of these inhibitors as potential therapeutics.

Abstract

RNA interference offers the potential of a novel therapeutic approach for treating skin disorders. To this end, we investigated delivery of nucleic acids, including a plasmid expressing the reporter gene luciferase, to mouse skin by intradermal injection into footpads using in vivo bioluminescence imaging over multiple time points. In order to evaluate the ability of RNA interference to inhibit skin gene expression, reporter gene constructs were co-injected with specific or non-specific siRNAs and the in vivo effects measured. Our results revealed that specific unmodified and modified siRNAs (but not nonspecific matched controls) strongly inhibit reporter gene expression in mice. These results indicate that small interfering RNA, delivered locally as RNA directly or expressed from viral or non-viral vectors, may be effective agents for treating skin disorders.

Abstract

Recruitment of CD4(+) T cells into islets is a critical component of islet inflammation (insulitis) leading to type 1 diabetes; therefore, determining if conditions used to treat diabetes change their trafficking patterns is relevant to the outcome. Cotransfer of CD4(+)BDC2.5 (BDC) cells with non-CD4 splenocytes obtained from newly diabetic NOD mice, but not when they are transferred alone, induces accelerated diabetes. It is unclear whether these splenocytes affect diabetes development by altering the systemic and/or local trafficking and proliferation patterns of BDC cells in target and nontarget tissues. To address these questions, we developed an animal model to visualize BDC cell trafficking and proliferation using whole-body in vivo bioluminescence imaging and used the images to direct tissue sampling for further analyses of the cell distribution within tissues. The whole-body, or macroscopic, trafficking patterns were not dramatically altered in both groups of recipient mice. However, the local patterns of cell distribution were distinct, which led to invasive insulitis only in cotransferred mice with an increased number of islet-infiltrating CD11b(+) and CD11c(+) cells. Taken together, the non-CD4 splenocytes act locally by promoting invasive insulitis without altering the systemic trafficking patterns or proliferation of BDC cells and thus contributing to diabetes by altering the localization within the tissue.

Abstract

Histologic interpretation of disease currently is performed with static images of excised tissues, and is limited by processing artifact, sampling error, and interpretive variability. The aim of this study was to show the use of functional optical imaging of viable mucosa for quantitative evaluation of colonic neoplasia in real time.Fluorescein (5 mg/mL) was administered topically in 54 human subjects undergoing screening colonoscopy. Fluorescence images were collected with 488-nm excitation at 12 frames/s with the confocal microendoscopy system. Movement of fluorescein in the transient period (<5 s) and the lamina propria:crypt contrast ratio in the steady-state phase (>5 s) were quantified.Normal mucosa showed circular crypts with uniform size, hyperplasia revealed proliferative glands with serrated lumens, and adenomas displayed distorted elongated glands. For t less than 5 seconds, fluorescein passed through normal epithelium with a peak speed of 1.14 +/- 0.09 microm/s at t = 0.5 seconds, and accumulated into lamina propria as points of fluorescence that moved through the interglandular space with an average speed of 41.7 +/- 3.4 microm/s. Passage of fluorescein through adenomatous mucosa was delayed substantially. For t greater than 5 seconds, high sensitivity, specificity, and accuracy was achieved using a discriminant function to evaluate the contrast ratio to distinguish normal from lesional mucosa (91%, 87%, and 89%, respectively; P < .001), hyperplasia from adenoma (97%, 96%, and 96%, respectively; P < .001), and tubular from villous adenoma (100%, 92%, and 93%, respectively; P < .001).Confocal imaging can be performed in vivo to assess the functional behavior of tissue in real time for providing pathologic interpretation, representing a new method for histologic evaluation.

Abstract

Treatment of small animals with radiation has in general been limited to planar fields shaped with lead blocks, complicating spatial localization of dose and treatment of deep-seated targets. In order to advance laboratory radiotherapy toward what is accomplished in the clinic, we have constructed a variable aperture collimator for use in shaping the beam of microCT scanner. This unit can image small animal subjects at high resolution, and is capable of delivering therapeutic doses in reasonable exposure times. The proposed collimator consists of two stages, each containing six trapezoidal brass blocks that move along a frame in a manner similar to a camera iris producing a hexagonal aperture of variable size. The two stages are offset by 30 degrees and adjusted for the divergence of the x-ray beam so as to produce a dodecagonal profile at isocenter. Slotted rotating driving plates are used to apply force to pins in the collimator blocks and effect collimator motion. This device has been investigated through both simulation and measurement. The collimator aperture size varied from 0 to 8.5 cm as the driving plate angle increased from 0 to 41 degrees. The torque required to adjust the collimator varied from 0.5 to 5 N x m, increasing with increasing driving plate angle. The transmission profiles produced by the scanner at isocenter exhibited a penumbra of approximately 10% of the collimator aperture width. Misalignment between the collimator assembly and the x-ray source could be identified on the transmission images and corrected by adjustment of the collimator location. This variable aperture collimator technology is therefore a feasible and flexible solution for adjustable shaping of radiation beams for use in small animal radiotherapy as well as other applications in which beam shaping is desired.

Abstract

Fourier transform infrared (FTIR) spectroscopy provides a unique molecular fingerprint of tissue from endogenous sources of light absorption; however, specific molecular components of the overall FTIR signature of precancer have not been characterized. In attenuated total reflectance mode, infrared light penetrates only a few microns of the tissue surface, and the influence of water on the spectra can be minimized, allowing for the analyses of the molecular composition of tissues. Here, spectra were collected from 98 excised specimens of the distal esophagus, including 38 squamous, 38 intestinal metaplasia (Barrett's), and 22 gastric, obtained endoscopically from 32 patients. We show that DNA, protein, glycogen, and glycoprotein comprise the principal sources of infrared absorption in the 950- to 1,800-cm(-1) regime. The concentrations of these biomolecules can be quantified by using a partial least-squares fit and used to classify disease states with high sensitivity, specificity, and accuracy. Moreover, use of FTIR to detect premalignant (dysplastic) mucosa results in a sensitivity, specificity, positive predictive value, and total accuracy of 92%, 80%, 92%, and 89%, respectively, and leads to a better interobserver agreement between two gastrointestinal pathologists for dysplasia (kappa = 0.72) versus histology alone (kappa = 0.52). Here, we demonstrate that the concentration of specific biomolecules can be determined from the FTIR spectra collected in attenuated total reflectance mode and can be used for predicting the underlying histopathology, which will contribute to the early detection and rapid staging of many diseases.

Abstract

To reveal, on a cellular and molecular level, how skeletal regeneration of a corticotomy is enhanced when using laser-plasma mediated ablation compared with conventional mechanical tissue removal.Osteotomies are well-known for their most detrimental side effect: thermal damage. This thermal and mechanical trauma to adjacent bone tissue can result in the untoward consequences of cell death and eventually in a delay in healing.Murine tibial corticotomies were performed using a conventional saw and a Ti:Sapphire plasma-generated laser that removes tissue with minimal thermal damage. Our analyses began 24 hours after injury and proceeded to postsurgical day 6. We investigated aspects of wound repair ranging from vascularization, inflammation, cell proliferation, differentiation, and bone remodeling.Histology of mouse corticotomy sites uncovered a significant difference in the onset of bone healing; whereas laser corticotomies showed abundant bone matrix deposition at postsurgical day 6, saw corticotomies only exhibited undifferentiated tissue. Our analyses uncovered that cutting bone with a saw caused denaturation of the collagen matrix due to thermal effects. This denatured collagen represented an unfavorable scaffold for subsequent osteoblast attachment, which in turn impeded deposition of a new bony matrix. The matrix degradation induced a prolonged inflammatory reaction at the cut edge to create a surface favorable for osteochondroprogenitor cell attachment. Laser corticotomies were absent of collagen denaturation, therefore osteochondroprogenitor cell attachment was enabled shortly after surgery.In summary, these data demonstrate that corticotomies performed with Ti:Sapphire lasers are associated with a reduced initial inflammatory response at the injury site leading to accelerated osteochondroprogenitor cell migration, attachment, differentiation, and eventually matrix deposition.

Abstract

Many therapeutic leads fail to advance clinically because of bioavailability, selectivity, and formulation problems. Molecular transporters can be used to address these problems. Molecular transporter conjugates of otherwise poorly soluble or poorly bioavailable drugs or probes exhibit excellent solubility in water and biological fluids and at the same time an enhanced ability to enter tissues and cells and with modification to do so selectively. For many conjugates, however, it is necessary to release the drug/probe cargo from the transporter after uptake to achieve activity. Here, we describe an imaging method that provides quantification of transporter conjugate uptake and cargo release in real-time in animal models. This method uses transgenic (luciferase) reporter mice and whole-body imaging, allowing noninvasive quantification of transporter conjugate uptake and probe (luciferin) release in real time. This process effectively emulates drug-conjugate delivery, drug release, and drug turnover by an intracellular target, providing a facile method to evaluate comparative uptake of new transporters and efficacy and selectivity of linker release as required for fundamental studies and therapeutic applications.

Abstract

A variety of viral-based and immune cell therapies have been proposed for use in the treatment of cancer. One possible approach to improve the effectiveness of these biological agents may be to combine them such that we can take advantage of natural immune cell-pathogen relationships. Here we discuss these potential approaches with particular emphasis on the use of immune cells as carrier vehicles to deliver viral therapies to the tumor.

Abstract

CD4(+)CD25(+) regulatory T cells (Tregs) suppress immune responses to alloantigens. The in vivo circulation and tissue localization of Tregs during an adaptive immune response remain unclear. We noninvasively tracked luciferase-expressing Tregs over time in an allogeneic bone marrow transplant model and demonstrated colocalization with effector T cells and initial expansion in secondary lymphoid organs before migration into inflamed tissues. Inflammation induced by irradiation and the allogeneic setting provided crucial stimuli for early Treg expansion and migration, leading to parallel reduction of effector T-cell proliferation in lymphoid organs and peripheral tissues. Treg transplants conferred long-term protection from systemic inflammatory challenge consistent with Treg in vivo survival. Suppression occurred during multiple phases of inflammation, but is optimal in the initial phase, providing protection from graft-versus-host disease while maintaining the graft-versus-tumor effect even at physiologic doses of Tregs due to their in vivo expansion, hence overcoming a major barrier to potential clinical applications of Tregs given their rarity.

Abstract

The first, to our knowledge, miniature dual-axes confocal microscope has been developed, with an outer diameter of 10 mm, for subsurface imaging of biological tissues with 5-7 microm resolution. Depth-resolved en face images are obtained at 30 frames per second, with a field of view of 800 x 100 microm, by employing a two-dimensional scanning microelectromechanical systems mirror. Reflectance and fluorescence images are obtained with a laser source at 785 nm, demonstrating the ability to perform real-time optical biopsy.

In vivo pathology: Seeing with molecular specificity and cellular resolution in the living bodyANNUAL REVIEW OF PATHOLOGY-MECHANISMS OF DISEASEContag, C. H.2007; 2: 277-305

Abstract

The emerging tools of in vivo molecular imaging are enabling dynamic cellular and molecular analyses of disease mechanisms in living animal models and humans. These advances have the potential to dramatically change a number of fields of study, including pathology, and to contribute to the development of regenerative medicine and stem cell therapies. The new tools of molecular imaging, which have already had a tremendous impact on preclinical studies, hold great promise for bringing important and novel information to the clinician and the patient. These approaches are likely to enable early diagnosis, rapid typing of molecular markers, immediate assessment of therapeutic outcome, and ready measures of the extent of tissue regeneration after damage. However, the full impact of these new techniques will be determined by our ability to translate them to the clinic and to develop a general strategy that integrates them with other advances in molecular diagnostics and molecular medicine.

Abstract

Due to the increased susceptibility of neonates to pathogens including those with mutations, the use of live vaccine strategies in the human population may present a potential risk to the young.The specific aim of this study was to assess the risk that prospective Salmonella enterica serovar Typhimurium vaccine strains pose for the neonate and determine whether the strains are an effective vaccine by assessing the adaptive immune response.To evaluate the susceptibility of young mice to potential vaccine strains, S. typhimuriumaroA(-) and Delta phoP mutant strains were labeled by chromosomal insertion of the lux operon--this serves as a readily traceable marker of infection using noninvasive imaging methods. BALB/c mice ages 1, 2, 4, and 6 weeks of age were fed the bioluminescent aroA(-) or Delta phoP strains and the course of infection was monitored by in vivobioluminescence imaging. In addition, blood samples were collected post-inoculation to assess the IgG response of mice to S. typhimurium LPS.Young BALB/c mice were not susceptible to the aroA(-) strain in contrast to their susceptibility to the Delta phoP strain at a dose of 10(9) colony forming units. Delivery by oral feeding of the aroA(-) and Delta phoP strains in young mice also produced a robust IgG anti-LPS response.Here, we report that young 2-week-old mice orally fed the bioluminescent aroA(-) S. typhimurium strain were not susceptible to infection and elicited a protective immune response.

Abstract

Bone marrow mononuclear cell (BMMC) therapy shows promise as a treatment for ischemic heart disease. However, the ability to monitor long-term cell fate remains limited. We hypothesized that molecular imaging could be used to track stem cell homing and survival after myocardial ischemia-reperfusion (I/R) injury. We first harvested donor BMMCs from adult male L2G85 transgenic mice constitutively expressing both firefly luciferase (Fluc) and enhanced green fluorescence protein reporter gene. Fluorescence-activated cell sorting analysis revealed approximately 0.07% of the population to consist of classic hematopoietic stem cells (lin-, thy-int, c-kit+, Sca-1+). Afterward, adult female FVB recipients (n = 38) were randomized to sham surgery or acute I/R injury. Animals in the sham (n = 16) and I/R (n = 22) groups received 5 x 10(6) of the L2G85-derived BMMCs via tail vein injection. Bioluminescence imaging (BLI) was used to track cell migration and survival in vivo for 4 weeks. BLI showed preferential homing of BMMCs to hearts with I/R injury compared with sham hearts within the first week following cell injection. Ex vivo analysis of explanted hearts by histology confirmed BLI imaging results, and quantitative real-time polymerase chain reaction (for the male Sry gene) further demonstrated a greater number of BMMCs in hearts with I/R injury compared with the sham group. Functional evaluation by echocardiography demonstrated a trend toward improved left ventricular fractional shortening in animals receiving BMMCs. Taken together, these data demonstrate that molecular imaging can be used to successfully track BMMC therapy in murine models of heart disease. Specifically, we have demonstrated that systemically delivered BMMCs preferentially home to and are retained by injured myocardium. Disclosure of potential conflicts of interest is found at the end of this article.

Abstract

Modern imaging technologies that allow for in vivo monitoring of cells in intact research subjects have opened up broad new areas of investigation. In the field of hematopoiesis and stem cell research, studies of cell trafficking involved in injury repair and hematopoietic engraftment have made great progress using these new tools. Multiple imaging modalities are available, each with its own advantages and disadvantages, depending on the specific application. For mouse models, clinically validated technologies such as magnetic resonance imaging (MRI) and positron emission tomography (PET) have been joined by optical imaging techniques such as in vivo bioluminescence imaging (BLI) and fluorescence imaging, and all have been used to monitor bone marrow and stem cells after transplantation into mice. Each modality requires that the cells of interest be marked with a distinct label that makes them uniquely visible using that technology. For each modality, there are several labels to choose from. Finally, multiple methods for applying these different labels are available. This chapter provides an overview of the imaging technologies and commonly used labels for each, as well as detailed protocols for gene delivery into hematopoietic cells for the purposes of applying these labels. The goal of this chapter is to provide adequate background information to allow the design and implementation of an experimental system for in vivo imaging in mice.

Abstract

Advances in imaging have enabled the study of cellular and molecular processes in the context of the living body that include cell migration patterns, location and extent of gene expression, degree of protein-protein interaction, and levels of enzyme activity. These tools, which operate over a range of scales, resolutions, and sensitivities, have opened up broad new areas of investigation where the influence of organ systems and functional circulation is intact. There are a myriad of imaging modalities available, each with its own advantages and disadvantages, depending on the specific application. Among these modalities, optical imaging techniques, including in vivo bioluminescence imaging and fluorescence imaging, use visible light to interrogate biology in the living body. Optimal imaging with these modalities require that the appropriate marker be used to tag the process of interest to make it uniquely visible using a particular imaging technology. For each optical modality, there are various labels to choose from that range from dyes that permit tissue contrast and dyes that can be activated by enzymatic activity, to gene-encoding proteins with optical signatures that can be engineered into specific biological processes. This article provides and overview of optical imaging technologies and commonly used labels, focusing on bioluminescence and fluorescence, and describes several examples of how these tools are applied to biological questions relating to the central nervous system.

Abstract

Heme oxygenase (HO), the rate-limiting enzyme in heme degradation, plays a role in neonatal jaundice. Understanding the regulation of the developmental expression patterns of the two HO isozymes, HO-1 and HO-2, is essential for targeting HO to control pathologic jaundice, and uncovering the fundamental role that they play in mammalian development. Here we characterized the ontogeny of HO-1 and HO-2 expression in the developing mouse cortex by in vivo bioluminescence imaging, quantitative RT-PCR, and Western blot. HO-2, the predominant isoform in the adult cortex, was relatively stable throughout all ages. HO-1 was observed to be progressively down-regulated in an age-related manner. HO-1 expression in the adult cortex was also the lowest among the eight adult tissues analyzed. Because there is a 283-bp CpG island region in the HO-1 promoter, we hypothesized that methylation of the island is responsible for the age-related HO-1 down-regulation in the cortex. Methylation status was assessed using regular and quantitative methylation-specific PCR and the CpG island was found to be hypomethylated at all ages. Therefore, we conclude that HO-1 gene expression in the cortex is developmentally-regulated and that methylation of the HO-1 CpG island is not associated with the down-regulation of the gene.

Abstract

In vivo bioluminescence imaging has become a cornerstone technology for preclinical molecular imaging. This imaging method is based on light-emitting enzymes, luciferases, which require specific substrates for light production. When linked to a specific biological process in an animal model of human biology or disease, the enzyme-substrate interactions become biological indicators that can be studied noninvasively in living animals. Signal intensity in these animal models depends on the availability of the substrate for the reaction within living cells in intact organs. The biodistribution and clearance rates of the substrates are therefore directly related to optimal imaging times and signal intensities and ultimately determine the sensitivity of detection and predictability of the model. Modifications of d-luciferin, the substrate for the luciferases obtained from beetle, including fireflies, result in novel properties and offer opportunities for improved bioassays. For this purpose, we have synthesized a conjugate, glycine-d-aminoluciferin, and investigated its properties relative to those of d-aminoluciferin and d-luciferin. The three substrates exhibited different kinetic properties and different intracellular accumulation profiles due to differences in their molecular structure, which in turn influenced their biodistribution in animals. Glycine-d-aminoluciferin had a longer in vivo circulation time than the other two substrates. The ability to assay luciferase in vitro and in vivo using these substrates, which exhibit different pharmacokinetic and pharmacodynamic properties, will provide flexibility and improve current imaging capabilities.

Abstract

A dual-axes confocal reflectance microscope has been developed that utilizes a narrowband laser at 1310 nm to achieve high axial resolution, image contrast, field of view, and tissue penetration for distinguishing among normal, hyperplastic, and dysplastic colonic mucosa ex vivo. Light is collected off-axis using a low numerical aperture objective to obtain vertical image sections, with 4- to 5-microm resolution, at tissue depths up to 610 microm. Post-objective scanning enables a large field of view (610 x 640 microm), and balanced-heterodyne detection provides sensitivity to collect vertical sections at one frame per second. System optics are optimized to effectively reject out-of-focus scattered light without use of a low-coherence gate. This design is scalable to millimeter dimensions, and the results demonstrate the potential for a miniature instrument to detect precancerous tissues, and hence to perform in vivo histopathology.

Abstract

Transient heating of tissues leading to cellular stress or death is very common in medicine and biology. In procedures involving a mild (below 70 degrees C) and prolonged (minutes) heating, such as hyperthermal tumor therapy, the cellular response to thermal stress is relatively well studied. However, there is practically no data on cell viability at higher temperatures and shorter exposures, while the demand for this knowledge is growing. Two main reasons motivate this research: (i) a growing number of laser therapies and surgical procedures involving pulsed heating, and (ii) cellular viability data at short exposures to high temperatures provide a unique insight into the understanding of processes leading to thermally induced cellular death. We designed a technique and performed a study of cell viability under pulses of heat from 0.3 to 100 ms in duration with peak temperatures as high as 130 degrees C. We found that the threshold of cellular death in this range can be accurately approximated by the Arrhenius law with the activation energy of 1 eV, a significantly lower value than was reported in studies based on multisecond exposures.

Abstract

Immunological reactions have a key role in health and disease and are complex events characterized by coordinated cell trafficking to specific locations throughout the body. Clarification of these cell-trafficking events is crucial for improving our understanding of how immune reactions are initiated, controlled and recalled. As we discuss here, an emerging modality for revealing cell trafficking is bioluminescence imaging, which harnesses the light-emitting properties of enzymes such as luciferase for quantification of cells and uses low-light imaging systems. This strategy could be useful for the study of a wide range of biological processes, such as the pathophysiology of graft-versus-host and graft-versus-leukaemia reactions.

Abstract

The design, synthesis, and evaluation of conjugates of arginine-rich transporters and luciferin are described that release luciferin only after entry into cells that are stably transfected with luciferase. Each molecule of free luciferin that is released after entry generates a photon that can be measured allowing for real-time quantification of uptake and release in cells. The process provides a method to assay uptake and release of free luciferin as a function of variations in the releasable linker and in the transporter.

Abstract

Delivery of therapeutics and imaging agents to target tissues requires localization and activation strategies with molecular specificity. Cell-associated proteases can be used for these purposes in a number of pathologic conditions, and their enzymatic activities can be exploited for activation strategies. Here, molecules based on the d-arginine octamer (r8) protein-transduction domain (PTD, also referred to as molecular transporters) have been adapted for selective uptake into cells only after proteolytic cleavage of a PTD-attenuating sequence by the prostate-specific antigen (PSA), an extracellular protease associated with the surface and microenvironment of certain prostate cancer cells. Convergent syntheses of these activatable PTDs (APTDs) are described, and the most effective r8 PTD-attenuating sequence is identified. The conjugates are shown to be stable in serum, cleaved by PSA, and taken up into Jurkat (human T cells) and PC3M prostate cancer cell lines only after cleavage by PSA. These APTD peptide-based molecules may facilitate targeted delivery of therapeutics or imaging agents to PSA-expressing prostate cancers.

Abstract

Some metalloporphyrins (Mps) inhibit heme oxygenase (HO), the rate-limiting enzyme in the production of bilirubin, and are potential compounds for the treatment of neonatal jaundice. We studied the safety and efficacy of Mps following oral administration. Adult HO-1-luc reporter mice were administered 30 micromol/kg body weight of tin mesoporphyrin (SnMP), zinc bis glycol deuteroporphyrin (ZnBG), or zinc protoporphyrin (ZnPP), or vehicle by oral gavage. Bilirubin production was measured as total body carbon monoxide (CO) excretion (VeCO). HO activity was quantitated via CO measurements by gas chromatography. HO-1 protein was determined by Western blot. HO-1 transcription levels were assessed by in vivo bioluminescence imaging. A significant 28% decrease in bilirubin production occurred within 3 h of SnMP treatment and persisted beyond 48 h. Bilirubin production decreased 15% and 9% by 3 h after administration of ZnBG and ZnPP, respectively, but returned to baseline within 48 h. Maximal inhibition of liver, spleen, and intestine HO activity was seen at 3 h with inhibitory effects decreasing in the order: SnMP > or = ZnBG > or = ZnPP. After SnMP treatment, HO-1 transcription increased 5.7-fold after 24 h. Furthermore, liver and spleen HO-1 protein significantly increased 3.7- and 2.0-fold, respectively, after 24 h. HO-1 transcription and protein were not affected in ZnBG- or ZnPP-treated mice. We conclude that the three Mps are absorbed at different rates in the mouse and affect bilirubin production and HO-1 expression in a tissue- and time-dependent manner.

Abstract

The development of effective cancer therapy will require the simultaneous targeting of multiple steps in tumor development. We have previously described an antiangiogenic gene therapy vector, Ad Flk1-Fc, which expresses a soluble VEGF receptor capable of inhibiting tumor angiogenesis and growth. We have also described an oncolytic virus, dl922/947, whose replication and subsequent cytotoxicity are restricted to cancer cells with a loss of the G1-S cell cycle checkpoint. Here we have optimized methods for combining these therapies, yielding significantly greater anti-tumor effects than the respective monotherapies. In cultured tumor lines, co-infection with both Ad Flk1-Fc and dl922/947 allowed replication and repackaging of the replication-deficient Ad Flk1-Fc and enhanced soluble VEGF receptor expression. Similar repackaging and increased gene expression were demonstrated in vivo using bioluminescence imaging studies. Finally, coadministration of these therapeutic viral therapies in vivo produced significantly enhanced anti-tumor effects in colon HCT 116 and prostate PC-3 xenografts in mice. This increased therapeutic benefit correlated with replication of Ad Flk1-Fc viral genomes, increased intratumoral levels of Flk1-Fc protein, and decreased microvessel density, consistent with enhanced antiangiogenic activity.

Abstract

Listeria monocytogenes is a ubiquitous gram-positive bacterium that can cause systemic and often life-threatening disease in immunocompromised hosts. This organism is largely an intracellular pathogen; however, we have determined that it can also grow extracellularly in animals, in the lumen of the gallbladder. The significance of growth in the gallbladder with respect to the pathogenesis and spread of listeriosis depends on the ability of the bacterium to leave this organ and be disseminated to other tissues and into the environment. Should this process be highly inefficient, growth in the gallbladder would have no impact on pathogenesis or spread, but if it occurs efficiently, bacterial growth in this organ may contribute to listeriosis and dissemination of this organism. Here, we use whole-body imaging to determine the efficacy and kinetics of food- and hormone-induced biliary excretion of L. monocytogenes from the murine gallbladder, demonstrating that transit through the bile duct into the intestine can occur within 5 min of induction of gallbladder contraction by food or cholecystokinin and that movement of bacteria through the intestinal lumen can occur very rapidly in the absence of fecal material. These studies demonstrate that L. monocytogenes bacteria replicating in the gallbladder can be expelled from the organ efficiently and that the released bacteria move into the intestinal tract, where they pass into the environment and may possibly reinfect the animal.

Abstract

Reductive prodrugs, mitomycin C and 5-aziridinyl-2,4-dinitrobenzamide (CB 1954), are nontoxic in their native form but become highly toxic upon reduction. Their effectiveness in cancer chemotherapy can be enhanced by delivering to tumors enzymes with improved prodrug reduction kinetics. We report the discovery of a new prodrug-reducing enzyme, YieF, from Escherichia coli, and the improvement of its kinetics for reducing mitomycin C and CB 1954. A YieF-derived enzyme, Y6, killed HeLa spinner cells with >or=5-fold efficiency than the wild-type enzymes, YieF and NfsA, at a variety of drug and enzyme concentrations and incubation times. With adhered HeLa cells and Salmonella typhimurium SL 7838 bacteria as enzyme delivery vehicle, at least an order of magnitude less of Y6-producing bacteria were required to kill >90% of tumor cells compared with bacteria expressing the wild-type enzymes, which at a comparable level killed < 5% of the cells. Thus, Y6 is a promising enzyme for use in cancer chemotherapy, and Salmonella strain SL 7838, which specifically targets tumors, may be used to deliver the prodrug-activating enzymes to tumors.

Abstract

The ability of small hairpin RNAs (shRNAs) to inhibit hepatitis C virus internal ribosome entry site (HCV IRES)-dependent gene expression was investigated in cultured cells and a mouse model. The results indicate that shRNAs, delivered as naked RNA or expressed from vectors, may be effective agents for the control of HCV and related viruses.

Abstract

Neonatal jaundice is commonly encountered and rarely associated with morbidity and mortality. Nonetheless, infants with glucose-6-phosphate dehydrogenase deficiency often have hemolysis (a heme load) caused by an environmental oxidant trigger, thus increasing their risk for serious morbidity. The use of tin mesoporphyrin (SnMP) has been proposed for interdicting the development of severe hyperbilirubinemia in a variety of conditions.We studied the in vivo effects of prophylactic oral SnMP on heme oxygenase (HO) activity and bilirubin production, as indexed by the excretion rate of carbon monoxide (VeCO), following a subsequent oral heme load.Adult mice were exposed serially to heme and assessed for in vivo bilirubin production rates, HO-1 transcription and protein, and HO activity. The effect of prophylaxis with a single oral dose of SnMP prior to an oral heme load was assessed by measuring VeCOand tissue HO activities.After serial heme exposures, VeCO, HO-1 transcription and protein, and liver and spleen HO activities increased incrementally. After pretreatment with oral SnMP, bilirubin production decreased in response to an oral heme load. Also, heme-mediated increases in liver, spleen, and intestine HO activities were significantly dampened.A single oral dose of SnMP results in durable inhibition of bilirubin production and HO activity for at least 24 h in a mouse model of oral heme loading. Further studies are needed to fully elucidate the duration of this protection against hyperbilirubinemia due to a delayed heme load and any long-term consequences of prophylaxis with SnMP on HO-1 transcription and HO-1 protein.

Abstract

Little is known about adoptive transfer of allogeneic ex vivo expanded dendritic cells (eDCs). We investigated the trafficking pattern of eDCs in mice after allogeneic bone marrow transplantation by using bioluminescence imaging. eDCs were expanded from bone marrow precursors in the presence of GM-CSF, interleukin-4, and Flt3L and retrovirally transduced to express luciferase (luc) and green fluorescence protein (gfp). Flow cytometry showed polyclonal DC populations after expansion that consisted of CD11c+CD11b+ and CD11c-CD11b+ cells that co-expressed CD40, CD80, CD86, and MHCII. eDCs were functional in mixed lymphocyte reactions and produced tumor necrosis factor-alpha on phytohemagglutinin stimulation. The eDCs were then injected intravenously into BALB/c recipient mice that had received allogeneic bone marrow transplantation 6 weeks previously. On day 1 after transfer, eDCs were detected by bioluminescence imaging throughout the lungs and spleen. In the later course, signals were observed throughout thymus, lower abdomen, and spleen throughout a period of more than 42 days. Immunofluorescence microscopy confirmed CD11c positivity on the gfp+ donor cells, which localized in T-cell zones of mesenteric lymph nodes, Peyer's patches, spleen, and thymus. These findings are important for adoptive immunotherapies because they indicate that eDCs migrate efficiently in vivo and are capable of surviving long term.

Abstract

Hemin upregulates heme oxygenase-1 (HO-1), a stress-induced enzyme implicated in protection from a variety of injuries while its related isoform HO-2 is constitutively expressed. The role of hemin or HO-1 in the pancreas and their potential modulation of pancreatic injury are unknown. We show that HO-1 is induced in pancreatitis caused by caerulein and more prominently in severe pancreatitis caused by feeding a choline-deficient diet (CDD). Intraperitoneal hemin administration dramatically increases peritoneal and pancreas macrophages that overexpress HO-1 in association with pancreatic induction of the chemoattractants monocyte chemotactic protein-1 and macrophage inflammatory protein-1alpha but not RANTES or macrophage inflammatory protein-2. Hemin administration before CDD feeding protected 8 of 8 mice from lethality while 7 of 16 controls died. Protection is mediated by HO-1-overexpressing macrophages since hemin-primed macrophages home to the pancreas after transfer to naive mice and protect from CDD-induced pancreatitis. Suppression of hemin-primed peritoneal cell HO-1 using HO-1-specific small interfering RNA prior to cell transfer abolishes protection from CDD-induced pancreatitis. Similarly, hemin pretreatment in caerulein-induced pancreatitis reduces serum amylase and lipase, decreases pancreatic trypsin generation, and protects from lung injury. Therefore, hemin-like compounds or hemin-activated macrophages may offer novel therapeutic approaches for preventing acute pancreatitis and its pulmonary complication via upregulation of HO-1.

Abstract

Treatment and prevention of hepatitis C virus (HCV) infections remain a major challenge for controlling this worldwide health problem; existing therapies are only partially effective and no vaccine is currently available. RNA interference offers the potential of a novel therapeutic approach for treating HCV infections. Toward this end, we evaluated small hairpin interfering RNAs (shRNAs) targeting the conserved internal ribosome entry site (IRES) element of the HCV genome for their ability to control gene expression in human cells and animals. We used a reporter gene plasmid in which firefly luciferase (fLuc) expression is dependent on the HCV IRES. Direct delivery of HCV IRES shRNAs efficiently blocked HCV IRES-mediated fLuc expression in transfected human 293FT cells as well as in a mouse model in which nucleic acids were delivered to liver cells by hydrodynamic transfection via the tail vein. These results indicate that shRNAs, delivered as RNA or expressed from viral or nonviral vectors, may be effective agents for the control of HCV and related viruses.

Abstract

We investigated the feasibility of bioluminescence imaging (BLI) for the in vivo assessment of cardiac allograft viability and visualization of passenger leukocytes during the course of acute rejection.Hearts of FVB (H-2q) luciferase-green fluorescent protein transgenic mice (beta-actin promoter) or FVB luciferase transgenic mice (CD5 promoter) were heterotopically transplanted into either BALB/c (H-2d) or FVB recipients. Light intensity emitting from the recipient animals was measured daily by in vivo BLI until 12 days after transplantation. Graft beating score (0 to 4) was assessed by daily abdominal palpation until 12 days after transplantation. Inflammatory cell infiltration (CD45 stain) and structural changes of green fluorescent protein-positive cardiomyocytes were followed by immunohistochemistry. All cardiac allografts were acutely rejected by 12 days after transplantation. The intensity of light emitting from cardiac allografts declined 4 days after transplantation and correlated with graft beating scores (R2=0.91, P=0.02). Immunohistochemistry confirmed these results by showing an increase of CD45+ inflammatory cell infiltration and destruction of green fluorescent protein-positive cardiomyocytes in the cardiac allografts during acute rejection. In vivo BLI visualized migration and proliferation of CD5+ passenger leukocytes in both syngeneic and allogeneic recipients. In the allograft recipients, light signal from CD5+ passenger leukocytes peaked at 6 hours and diminished by 12 hours, whereas in the syngeneic recipients, the signal remained high until 10 days after transplantation.BLI is a useful modality for the quantitative assessment of in vivo cardiac graft viability and tracking of passenger leukocytes in vivo during the course of acute rejection.

Abstract

A novel application of bioluminescence for nucleic acid quantification, the bioluminescence regenerative cycle (BRC), is described in theoretical terms and supported by preliminary experimental data. In the BRC system, pyrophosphate (PPi) molecules are released during biopolymerization and are counted and correlated to DNA copy number. The enzymes ATP-sulfurylase and firefly luciferase are employed to generate photons quantitatively from PPi. Enzymatic unity-gain positive feedback is implemented to amplify photon generation and to compensate for decay in light intensity by self-regulation. The cumulative total of photons can be orders of magnitude higher than in typical chemiluminescent processes. A system level theoretical model is developed, taking into account the kinetics of the regenerative cycle, contamination, and detector noise. Data and simulations show that the photon generation process achieves steady state for the time range of experimental measurements. Based on chain reaction theory, computations show that BRC is very sensitive to variations in the efficiencies of the chemical reactions involved and less sensitive to variations in the quantum yield of the process. We show that BRC can detect attomolar quantities of DNA (10(-18) mol), and that the useful dynamic range is five orders of magnitude. Sensitivity is not constrained by detector performance but rather by background bioluminescence caused by contamination by either PPi or ATP (adenosine triphosphate).

Abstract

Graft-versus-host disease (GVHD) is a major obstacle in allogeneic hematopoietic cell transplantation. Given the dynamic changes in immune cell subsets and tissue organization, which occur in GVHD, localization and timing of critical immunological events in vivo may reveal basic pathogenic mechanisms. To this end, we transplanted luciferase-labeled allogeneic splenocytes and monitored tissue distribution by in vivo bioluminescence imaging. High-resolution analyses showed initial proliferation of donor CD4+ T cells followed by CD8+ T cells in secondary lymphoid organs with subsequent homing to the intestines, liver, and skin. Transplantation of purified naive T cells caused GVHD that was initiated in secondary lymphoid organs followed by target organ manifestation in gut, liver, and skin. In contrast, transplanted CD4+ effector memory T (T(EM)) cells did not proliferate in secondary lymphoid organs in vivo and despite their in vitro alloreactivity in mixed leukocyte reaction (MLR) assays did not cause acute GVHD. These findings underline the potential of T-cell subsets with defined trafficking patterns for immune reconstitution without the risk of GVHD.

Abstract

Tissue regeneration and transplantation of solid organs involve complex processes that can only be studied in the context of the living organism, and methods of analyzing these processes in vivo are essential for development of effective transplantation and regeneration procedures. We utilized in vivo bioluminescence imaging (BLI) to noninvasively visualize engraftment, survival, and rejection of transplanted tissues from a transgenic donor mouse that constitutively expresses luciferase. Dynamic early events of hematopoietic reconstitution were accessible and engraftment from as few as 200 transplanted whole bone marrow (BM) cells resulted in bioluminescent foci in lethally irradiated, syngeneic recipients. The transplantation of autologous pancreatic Langerhans islets and of allogeneic heart revealed the tempo of transplant degeneration or immune rejection over time. This imaging approach is sensitive and reproducible, permits study of the dynamic range of the entire process of transplantation, and will greatly enhance studies across various disciplines involving transplantation.

Abstract

Neonates and young children are acutely susceptible to infections by gastrointestinal bacterial pathogens, such as Salmonella enterica serovar Typhimurium (S. typhimurium). To reveal age-related differences in susceptibility to this pathogen, we used in vivo bioluminescence imaging (BLI) to monitor the progression of infection in neonatal (1-wk-old), suckling (2-wk-old), juvenile (4-wk-old), and adult (6-wk-old) BALB/c mice. Mice were orally infected with various doses of a bioluminescent-labeled wild-type or mutant S. typhimurium strain, and progression of infection was monitored by BLI for 2 wks. We found that neonatal and suckling mice were more susceptible to the wild-type strain at inoculum sizes 4 and 2 log(10)'s lower for neonatal and suckling mice, respectively, than those for adult mice. At the lower inocula, newborn mice showed disseminated systemic infection as indicated by the pattern of photon emission assessed by BLI, whereas no bioluminescent signals were detectable in adult mice. In addition, an orgA(-) mutant strain of S. typhimurium with reduced virulence in adult mice produced systemic infection in newborn, suckling, and juvenile mice. Furthermore, as low as 3 log(10) CFU could be detected by BLI in tissue. The present study demonstrates that susceptibility to S. typhimurium infection decreases with age. Also, we established that BLI can be used to monitor the progression of infection in mice. Thus, this model of age-related susceptibility to S. typhimurium using BLI can be used to advance our understanding of the mechanisms involved in newborn susceptibility to infection.

Abstract

Smad2 and Smad3 (Smad2/3) proteins are key signaling molecules for TGF-beta and some related family members regulating the transcription of several hundred genes. TGF-beta have key roles in development, tissue homeostasis, and the pathogenesis of many human diseases, including cancer, fibrotic disorders, developmental defects, and neurodegeneration. To study the temporal and spatial patterns of Smad2/3-dependent signaling in normal and pathological conditions in the living organism, we engineered transgenic mice with a Smad-responsive luciferase reporter construct (SBE-luc mice). Using bioluminescent imaging, we assessed Smad2/3 signaling activity noninvasively in living mice. At baseline, this activity was highest in brain, intestine, heart, and skin, and correlated with biochemical measurements of reporter activity. Primary astrocytes cultured from SBE-luc mice showed specific activation of the reporter in response to Smad2/3-activating TGF-beta family members. Treatment of mice with the endotoxin LPS resulted in a fast and vigorous, but transient activation of the reporter in the intestine. Although the response was similarly rapid in brain, it remained increased, indicating important but different cellular responses to endotoxin challenge in these organs. Traumatic brain injury with a needle stab resulted in local activation of Smad2/3-dependent genes and a severalfold increase in bioluminescence in living mice. SBE-luc mice can therefore be used to study temporal, tissue-specific activation of Smad2/3-dependent signaling in living mice in normal or pathological conditions as well as for the identification of endogenous or synthetic modulators of this pathway.

Abstract

Previous studies have identified Sleeping Beauty transposons as efficient vectors for nonviral gene delivery in mammalian cells. However, studies demonstrating the usefulness of transposons as gene delivery vehicles into adult stem cells are lacking. Multipotent adult progenitor cells (MAPC) are nonhematopoietic stem cells with the capacity to form most, if not all, cell types of the body and as such hold great therapeutic potential. The whole-body biodistribution and persistence of MAPC are unknown, and such data would help direct clinical applications. We have nucleofected murine MAPC with two plasmid-based Sleeping Beauty transposons encoding the red fluorescent protein (DsRed2) and firefly luciferase. Transgenic euploid MAPC clones maintained their characteristic multilineage differentiation potential in vitro. DsRed2 and luciferase expression allowed for MAPC detection in vivo and in tissue sections. To confirm that transgenesis occurred by transposition into the genome of MAPC, we mapped Sleeping Beauty transposon integration sites in two MAPC clones using splinkerette PCR. This novel dual-reporter imaging approach based on the transgenesis of MAPC with Sleeping Beauty transposons sheds light on the homing patterns of MAPC and paves the way for quantification of MAPC engraftment in real time in vivo.

Abstract

In vivo bioluminescence imaging depends on light emitted by luciferases in the body overcoming the effect of tissue attenuation. Understanding this relationship is essential for detection and quantification of signal. We have studied four codon optimized luciferases with different emission spectra, including enzymes from firefly (FLuc), click beetle (CBGr68, CBRed) and Renilla reniformins (hRLuc). At 25 degrees C, the in vitro lambda(max) of these reporters are 578, 543, 615, and 480 nm, respectively; at body temperature, 37 degrees C, the brightness increases and the firefly enzyme demonstrates a 34-nm spectral red shift. Spectral shifts and attenuation due to tissue effects were evaluated using a series of 20-nm bandpass filters and a cooled charge-coupled device (CCD) camera. Attenuation increased and the spectra of emitted light was red shifted for signals originating from deeper within the body relative to superficial origins. The tissue attenuation of signals from CBGr68 and hRLuc was greater than from those of Fluc and CBRed. To further probe tissue effects, broad spectral emitters were created through gene fusions between CBGr68 and CBRed. These resulted in enzymes with broader emission spectra, featuring two peaks whose intensities are differentially affected by temperature and tissue depth. These spectral measurement data allow for improved understanding of how these reporters can be used in vivo and what they can reveal about biological processes in living subjects.

Abstract

Mast cells are important effector cells in IgE-associated immune responses, but also can contribute to host defense in certain examples of bacterial infection. We found that genetically mast cell-deficient WBB6F1-Kit(W)/Kit(W-v) mice exhibited more bacterial CFUs per spleen by 6 days after intraperitoneal injection of bioluminescent Salmonella typhimurium, and died more rapidly after infection, than did the congenic WBB6F1-Kit(+/+) wild type mice. Adoptive transfer of bone marrow-derived cultured mast cells of Kit(+/+) origin to the peritoneal cavity of Kit(W)/Kit(W-v) mice resulted in engraftment of mast cells in the peritoneal cavity and mesentery of the recipient mice, and the development of large numbers of mast cells in the spleen. However, such mast cell-engrafted Kit(W)/Kit(W-v) mice appeared sicker after intraperitoneal injection with S. typhimurium than did mast cell-deficient Kit(W)/Kit(W-v) mice, and exhibited numbers of CFUs of bacteria per spleen, and a survival curve, that were not significantly different than those of Kit(W)/Kit(W-v) mice. These results, when taken together with prior studies investigating the roles of mast cells in innate immunity, strongly suggest that whether mast cells can be shown to have a significant role in enhancing survival during bacterial infections may depend critically on the details of the particular experimental systems examined.

Abstract

To identify novel treatments for pediatric solid tumors and/or for malignancies with low-level Her2/neu expression.Using fluorescence-activated cell sorting and immunohistochemistry, Her2/neu expression was determined on cell lines derived vfrom Ewing's family tumors (EFT) and neuroblastoma. Sensitivity to trastuzumab treatment was investigated using an in vitro proliferation assay. Cytotoxicity against EFT cell lines was done with either freshly isolated or ex vivo activated and expanded T cells (cytokine-induced killer cells, CIK cells), with or without addition of a CD3xHer2/neu bispecific antibody. The effects of either trastuzumab, CIK cells alone, or CD3xHer2/neu bispecific antibody redirected CIK cells was determined using a SCID/hu model of EFTs and serial, noninvasive bioluminescent imaging.EFT cell lines express 5- to 10-fold lower levels of her2/neu than either breast (BT-474) or ovarian (SK-OV-3) cell lines. Treatment of EFT cell lines with trastuzumab did not induce growth inhibition either in vitro or in vivo. In contrast, Her2/neu could be used to redirect CIK cell to mediate cytotoxicity against EFTs both in vitro and in vivo (using two different treatment schemas).CD3xHer2/neu bispecific antibody and CIK cells may be a suitable approach to treat malignancies with low-level Her2/neu expression not responsive to trastuzumab.

Abstract

Reconstruction of craniofacial defects presents a substantial biomedical burden, and requires complex surgery. Interestingly, children after age 2 years and adults are unable to heal large skull defects. This nonhealing paradigm provides an excellent model system for craniofacial skeletal tissueengineering strategies. Previous studies have documented the in vivo osteogenic potential of adipose-derived stromal (ADS) cells and bone marrow-derived stromal (BMS) cells. This study investigates the ability to accelerate in vivo osteogenesis on ex vivo recombinant human bone morphogenetic protein 2 (BMP-2) and retinoic acid stimulation. Mouse osteoblasts, ADS cells, and BMS cells were seeded onto apatite-coated PLGA scaffolds, stimulated with rhBMP-2 and retinoic acid ex vivo for 4 weeks, and subsequently implanted into critically sized (4 mm) calvarial defects. Samples were harvested after 2, 4, 8, and 12 weeks. Areas of complete bony bridging were noted as early as 2 weeks in vivo; however, osteoclasts were attracted to the scaffold as identified by calcitonin receptor staining and tartrate-resistant acid phosphatase activity staining. Although the optimal method of in vitro osteogenic priming for mesenchymal cells remains unknown, these results provide evidence that BMP-2 and retinoic acid stimulation of multipotent cells ex vivo can subsequently induce significant quantities of bone formation within a short time period in vivo.

Abstract

Direct radiolabeling of proteins can result in the loss of targeting activity, requires highly customized procedures, and yields heterogeneous products. Here we describe a novel imaging complex comprised of a standardized (99m)Tc-radiolabeled adapter protein noncovalently bound to a "Docking tag" fused to a "Targeting protein". The assembly of this complex is based on interactions between human 109-amino acid (HuS) and 15-amino acid (Hu-tag) fragments of ribonuclease I, which serve as an "Adapter protein" and a Docking tag, respectively.HuS modified with hydrazinonicotinamide (HYNIC) was radiolabeled using (99m)Tc-tricine to a specific activity of 3.4-7.4 MBq/microg. Protein complexes were then formed by mixing (99m)Tc-HuS with equimolar amounts of either Hu-tagged VEGF(121) (Hu-VEGF [vascular endothelial growth factor]) or Hu-tagged anti-VEGFR-2 single-chain antibody (Hu-P4G7) and incubating on ice for 15 min. 4T1 luc/gfp luciferase-expressing murine mammary adenocarcinoma cells (1 x 10(4)) were implanted subcutaneously or injected intravenously into BALB/c mice. Bioluminescent imaging (BLI) was performed 10 d later. Immediately after BLI visualization of tumor, 18.5-37 MBq of tracer (5-10 microg of protein) were injected via tail vein. One hour later planar or SPECT images were obtained, followed by killing the mice.There was significantly (P = 0.0128) increased uptake of (99m)Tc-HuS/Hu-VEGF (n = 10) within subcutaneous tumor as compared with (99m)Tc-HuS/Hu-P4G7 (n = 5) at biodistribution assay (2.68 +/- 0.75 vs. 1.8 +/- 0.21; tumor-to-subcutaneous tissue [ratio of specific activities], respectively), despite similar molecular weights. The focal (99m)Tc-HuS/Hu-VEGF uptake seen on planar images (3.44 +/- 1.16 [tumor to soft-tissue background]) corresponded directly to the locations of tumor observed by BLI. Region of interest analyses of SPECT images revealed a significant increase of (99m)Tc-HuS/Hu-VEGF (n = 5) within the lungs with BLI-detectable pulmonary tumor nodules as compared with controls (n = 4) (right: 4.47 +/- 2.07 vs. 1.79 +/- 0.56; left: 3.66 +/- 1.65 vs. 1.62 +/- 0.45, tumor lung [counts/pixel]/normal lung [counts/pixel], respectively).(99m)Tc-HuS/Hu-VEGF complex is stable for at least 1 h in vivo and can be effectively used to image mouse tumor neovasculature in lesions as small as several millimeters in soft tissue. We expect that a similar approach can be adapted for in vivo delivery of other targeting proteins of interest without affecting their bioactivity.

Abstract

We present a novel confocal microscope that has dual-axis architecture and biaxial postobjective scanning for the collection of fluorescence images from biological specimens. This design uses two low-numerical-aperture lenses to achieve high axial resolution and long working distance, and the scanning mirror located distal to the lenses rotates along the orthogonal axes to produce arc-surface images over a large field of view (FOV). With fiber optic coupling, this microscope can potentially be scaled down to millimeter dimensions via microelectromechanical systems (MEMS) technology. We demonstrate a benchtop prototype with a spatial resolution < or =4.4 microm that collects fluorescence images with a high SNR and a good contrast ratio from specimens expressing GFP. Furthermore, the scanning mechanism produces only small differences in aberrations over the image FOV. These results demonstrate proof of concept of the dual-axis confocal architecture for in vivo molecular and cellular imaging.

Abstract

Craniosynostosis, the premature fusion of cranial sutures, is one of the most common craniofacial anomalies, with a reported incidence of up to one in 2500 live births. Despite its prevalence, the cause of craniosynostosis remains unknown. Previously, apoptosis has been postulated to be a contributing factor in the pathogenesis of craniosynostosis, although the role of programmed cell death in cranial sutures is poorly understood. To address this problem, the authors used an established rodent model of posterior-frontal suture fusion and sagittal suture patency to globally examine apoptosis in cranial sutures. Apoptosis was evaluated by systemically coinjecting Sprague-Dawley rats with both fluorescent and technetium-99m-labeled annexin V at time points before, during, and after the period of predicted posterior-frontal suture fusion to determine the magnitude and time course of overall apoptotic activity in both fusing and patent sutures. Using these novel in situ imaging techniques, the authors observed a significant increase in the overall levels of apoptosis in both the posterior-frontal and sagittal suture complexes during the period of predicted posterior-frontal suture fusion. To further explore this increase in apoptotic activity, they used microarray technology to study apoptosis-related genes within the suture complex. Interestingly, there was activation of distinct apoptotic pathways in the posterior-frontal and sagittal sutures during the period of predicted posterior-frontal suture fusion. Whereas increased transcription of genes associated with the mitochondria-mediated apoptotic pathway occurred in the posterior-frontal suture during fusion, activation of genes associated with the death receptor-mediated apoptotic pathway predominated in the patent sagittal suture during the same time period. These data suggest that although overall apoptotic activity in rat patent and fusing sutures is similar, the pathways mediating apoptosis within each suture are distinct.

Abstract

In adults and children over two years of age, large cranial defects do not reossify successfully, posing a substantial biomedical burden. The osteogenic potential of bone marrow stromal (BMS) cells has been documented. This study investigates the in vivo osteogenic capability of adipose-derived adult stromal (ADAS) cells, BMS cells, calvarial-derived osteoblasts and dura mater cells to heal critical-size mouse calvarial defects. Implanted, apatite-coated, PLGA scaffolds seeded with ADAS or BMS cells produced significant intramembranous bone formation by 2 weeks and areas of complete bony bridging by 12 weeks as shown by X-ray analysis, histology and live micromolecular imaging. The contribution of implanted cells to new bone formation was 84-99% by chromosomal detection. These data show that ADAS cells heal critical-size skeletal defects without genetic manipulation or the addition of exogenous growth factors.

Abstract

Understanding biological processes in the context of intact organ systems with fine temporal resolution has required the development of imaging strategies that reveal cellular and molecular changes in the living body. Reporter genes that confer optical signatures on a given biological process have been used widely in cell biology and have been used more recently to interrogate biological processes in living animal models of human biology and disease. The use of internal biological sources of light, luciferases, to tag cells, pathogens, and genes has proved to be a versatile tool to provide in vivo indicators that can be detected externally. The application of this technology to the study of animal models of infectious disease has not only provided insights into disease processes, but has also revealed new mechanisms by which pathogens may avoid host defences during infection.

Abstract

The expression of CD5 increases progressively as thymocytes mature. We have shown that CD5 expression is controlled by a tissue-specific regulatory promoter located upstream of the CD5 translation start sites. Deletion of this regulatory promoter, which contains three potential transcription factor binding sites (CCAAT, kappa E2, and ets) reduces the promoter activity to basal level. Of these sites, only ets proved essential for CD5 expression in T cell lines. Here, we introduce a role for the E47 transcription factor and the CD5 promoter kappa E2 site in regulating CD5 expression during thymocyte development. Using T cell lines, we show that (i) mutation of the kappa E2 site in the CD5 regulatory promoter results in a significant elevation of CD5 promoter activity; (ii) the E47 transcription factor binds to the kappa E2 site; and (iii) overexpression of E47 inhibits CD5 expression. We then show, in high-dimensional fluorescence-activated cell sorting studies with primary thymocytes at successive developmental stages, that (i) intracellular E47 levels decrease as surface CD5 expression increases; (ii) E47 expression is down-regulated and CD5 expression is correspondingly up-regulated in DN3 thymocytes in RAG-2-deficient mice injected with anti-CD3 to mimic pre-T cell receptor stimulation; and (iii) E47 expression is down-regulated and CD5 expression is up-regulated when double positive thymocytes are stimulated in vitro with anti-CD3. Based on these data, we propose that E47 negatively regulates CD5 expression by interacting with the kappa E2 site in the CD5 regulatory promoter and that decreases in E47 in response to developmental signals are critical to the progressive increase in CD5 expression as thymocytes mature.

Abstract

We define five unique cellular responses to thermal stress using a reporter construct generated using the stress-inducible promoter from the gene encoding a murine 70 kDa heat shock protein (Hsp70A.1) to express luciferase (luc). Thermal stress was delivered over a range of temperatures (42-68 degrees C) for 5 s to 20 min and luciferase activity was measured in live cells using a cooled CCD camera as a measure of reporter gene transcription. Reporter gene expression was assessed every 2 h for 10 h, and at 24 h post-stress. Expression patterns were validated for selected temperatures. A transition zone where cells lose the ability to produce light and beyond which >50% of cells die was observed to occur within a narrow (2.5 degrees C) temperature window. Although luc and hsp70 mRNA levels in this transition zone were high, there were reduced levels of Luc and Hsp70 protein and ATP levels. Cells treated at these temperatures recovered the ability to produce light in response to a secondary stress at 30 h. This Hsp70-luc reporter gene construct may be useful for defining zones of physiologic responses and assessing collateral thermal damage generated during treatment of biological tissue with lasers and other sources of heat.

Abstract

We examined the role of redox signaling generated by NADPH oxidase in activation of NF-kappaB and host defense against Pseudomonas aeruginosa pneumonia. Using mice with an NF-kappaB-driven luciferase reporter construct (HIV-LTR/luciferase (HLL)), we found that intratracheal administration of P. aeruginosa resulted in a dose-dependent neutrophilic influx and activation of NF-kappaB. To determine the effects of reactive oxygen species generated by the NADPH oxidase system on activation of NF-kappaB, we crossbred mice deficient in p47(phox) with NF-kappaB reporter mice (p47(phox-/-)HLL). These p47(phox-/-)HLL mice were unable to activate NF-kappaB to the same degree as HLL mice with intact NADPH oxidase following P. aeruginosa infection. In addition, lung TNF-alpha levels were significantly lower in p47(phox-/-)HLL mice compared with HLL mice. Bacterial clearance was impaired in p47(phox-/-)HLL mice. In vitro studies using bone marrow-derived macrophages showed that Toll-like receptor 4 was necessary for NF-kappaB activation following treatment with P. aeruginosa. Additional studies with macrophages from p47(phox-/-) mice confirmed that redox signaling was necessary for maximal Toll-like receptor 4-dependent NF-kappaB activation in this model. These data indicate that the NADPH oxidase-dependent respiratory burst stimulated by Pseudomonas infection contributes to host defense by modulating redox-dependent signaling through the NF-kappaB pathway.

Abstract

Radiolabeled annexin V may provide an early indication of the success or failure of anticancer therapy on a patient-by-patient basis as an in vivo marker of tumor cell killing. An important question that remains is when, after initiation of treatment, should annexin V imaging be performed. To address this issue, we obtained simultaneous in vivo measurements of tumor burden and uptake of radiolabeled annexin V in the syngeneic orthotopic murine BCL1 lymphoma model using in vivo bioluminescence imaging (BLI) and small animal single-photon emission computed tomography (SPECT). BCL1 cells labeled for fluorescence and bioluminescence assays (BCL1-gfp/luc) were injected into mice at a dose that leads to progressive disease within two to three weeks. Tumor response was followed by BLI and SPECT before and after treatment with a single dose of 10 mg/kg doxorubicin. Biodistribution analyses revealed a biphasic increase of annexin V uptake within the tumor-bearing tissues of mice. An early peak occurring before actual tumor cells loss was observed between 1 and 5 hr after treatment, and a second longer sustained rise from 9 to 24 hr after therapy, which heralds the onset of tumor cell loss as confirmed by BLI. Multimodality imaging revealed the temporal patterns of tumor cell loss and annexin V uptake revealing a better understanding of the timing of radiolabeled annexin V uptake for its development as a marker of therapeutic efficacy.

Abstract

Assessment of laser-induced tissue damage is not complete without an investigation into the resulting cellular and molecular changes. In the past, tissue damage was quantified macroscopically by visual effects such as tissue mass removal, carbonization and melting. Microscopically, assessment of tissue damage has been typically limited to histological analysis of excised tissue samples. In this research, we used heat shock protein (hsp70) transcription to track cellular response to laser-induced injury. A stable cell line (NIH-3T3) was generated containing the firefly luciferase (luc) reporter gene attached to the hsp promoter (murine hsp70a1). After thermal injury with a pulsed holmium-yttrium aluminum garnet laser (lambda = 2.1 microm, taup = 250 micros, 30 pulses, 3 Hz), luciferase is produced on hsp70 activation and emits broad-spectrum bioluminescence over a range of 500-700 nm, with a peak at 563 nm. The onset of bioluminescence can be seen as early as 2 h after treatment and usually peaks at 8-12 h depending on the severity of heat shock. The luminescence was quantified in live cells using bioluminescence imaging. A minimum pulse energy (65 mJ/pulse [total energy 1.95 J; total radiant exposure = 6 J/cm2]) was needed to activate the hsp70 response, and a higher energy (103 mJ/pulse [total energy 3.09 J; total radiant exposure = 9.6 J/cm2]) was associated with a reduction in hsp70 response and cell death. Bioluminescence levels correlated well with actual hsp70 protein concentrations as determined by enzyme-linked immunosorbent assay. Photon counts were normalized to the percentage of live cells by means of a flow cytometry cell viability assay. Within a relatively small range between a lower activation threshold and an upper threshold that leads to cell death, the hsp70 response followed an Arrhenius relationship when constant-temperature water bath and laser experiments were carried out.

Abstract

In vivo imaging of bioluminescent reporters relies on expression of light-emitting enzymes, luciferases, and delivery of chemical substrates to expressing cells. Coelenterazine (CLZN) is the substrate for a group of bioluminescent enzymes obtained from marine organisms. At present, there are more than 10 commercially available CLZN analogs. To determine which analog is most suitable for activity measurements in live cells and living animals, we characterized 10 CLZN analogs using Renilla luciferase (Rluc) as the reporter enzyme. For each analog, we monitored enzyme activity, auto-oxidation, and efficiency of cellular uptake. All CLZN analogs tested showed higher auto-oxidation signals in serum than was observed in phosphate buffer or medium, mainly as a result of auto-oxidation by binding to albumin. CLZN-f, -h, and -e analogs showed 4- to 8-fold greater Rluc activity, relative to CLZN-native, in cells expressing the enzyme from a stable integrant. In studies using living mice expressing Rluc in hepatocytes, administration of CLZN-e and -native produced the highest signal. Furthermore, distinct temporal differences in signal for each analog were revealed following intravenous or intraperitoneal delivery. We conclude that the CLZN analogs that are presently available vary with respect to hRluc utilization in culture and in vivo, and that the effective use of CLZN-utilizing enzymes in living animals depends on the selection of an appropriate substrate.

Abstract

We present a dual-axes confocal microscope that employs postobjective scanning and low-coherence heterodyne detection to collect vertical cross-sectional images from biological tissue with high axial resolution, reduced noise from scattered light, deep tissue penetration, and a large dynamic range. This architecture can be scaled down to millimeter dimensions with microelectromechanical systems technology for performance of in vivo optical biopsy.

Abstract

The effector functions of immune cells have typically been examined using assays that require sampling of tissues or cells to reveal specific aspects of an immune response (e.g., antigen-specificity, cytokine expression or killing of target cells). The outcome of an immune response in vivo, however, is not solely determined by a single effector function of a specific cell population, but is the result of numerous cellular and molecular interactions that occur in the complex environment of intact organ systems. These interactions influence survival, migration, and activation, as well as final effector function of a given population of cells. Efforts to reveal the cellular and molecular basis of biological processes have resulted in a number of technologies that combine molecular biology and imaging sciences that are collectively termed as Molecular Imaging. This emerging field has developed to reveal functional aspects of cells, genes, and proteins in real time in living animals and humans and embraces multiple modalities, including established clinical imaging methods such as magnetic resonance imaging, single photon emission computed tomography, and positron emission tomography, as well as novel methodologies specifically designed for research animals. Here, we highlight one of the newer modalities, in vivo bioluminescence imaging, as a method for evaluating effector T cell proliferation, migration, and function in model systems of malignant and non-malignant diseases.

Abstract

The lipid bilayer of a cell presents a significant barrier for the delivery of many molecular imaging reagents into cells at target sites in the body. Protein translocation domains (PTDs) are peptides that breach this barrier. Conjugation of PTDs to imaging agents can be utilized to facilitate the delivery of these agents through the cell wall, and in some cases, into the cell nucleus, and have potential for in vitro and in vivo applications. PTD imaging conjugates have included small molecules, peptides, proteins, DNA, metal chelates, and magnetic nanoparticles. The full potential of the use of PTDs in novel in vivo molecular probes is currently under investigation. Cells have been labeled in culture using magnetic nanoparticles derivatized with a PTD and monitored in vivo to assess trafficking patterns relative to cells expressing a target antigen. In vivo imaging of PTD-mediated gene transfer to cells of the skin has been demonstrated in living animals. Here we review several natural and synthetic PTDs that have evolved in the quest for easier translocation across biological barriers and the application of these peptide domains to in vivo delivery of imaging agents.

Abstract

Nonviral gene transfer offers biosafety, stability, and expense advantages over viruses; however, it has suffered from poor efficiency. Because arginine-rich peptides facilitate uptake of macromolecules such as proteins, liposomes, and iron nanoparticles, we explored their potential in enhancing plasmid DNA delivery. In their unmodified form, known protein transduction sequences, including hepta-arginine and Tat(47-57), failed to support effective gene delivery. However, by flanking a core of consecutive arginines with amino- and carboxy-terminal cysteines in vitro gene transfer was observed. Furthermore, interspersing arginines with glycine and histidine residues achieved reversible plasmid condensation and dramatically increased transfection levels in a variety of cell types. Unlike most available cationic homopolymers that function only in vitro, these new peptides also increased gene expression in both murine and human tissue in vivo. Thus, cysteine-flanked, internally spaced arginine-rich (CFIS-R) peptides represent a new approach to efficient nonviral plasmid delivery using rationally designed protein transduction domains.

Abstract

Hepatitis C virus (HCV) is an RNA virus infecting one in every 40 people worldwide. Current treatments are ineffective and HCV is the leading cause of liver failure leading to transplantation in the United States and Europe. Translational control of HCV is a prime therapeutic target. We assessed the inhibitory potential of morpholino phosphoramidate antisense oligonucleotides (morpholinos) on HCV translation by codelivering them with reporter plasmids expressing firefly luciferase under the translational control of the HCV internal ribosome entry site (IRES) into the livers of mice. Real-time imaging of HCV IRES luciferase reporter messenger RNA (mRNA) translation in living mice showed that a 20-mer complementary to nucleotides 345-365 of the IRES inhibited translation by greater than 95% for at least 6 days and showed mismatch specificity. No significant nonspecific inhibition of a cap-dependent luciferase or encephalomyocarditis virus (EMCV) IRES luciferase reporter translation was observed. Inhibition by the 20-mer morpholino was dose dependent, with 1 nmol/mouse giving the highest inhibition. In conclusion, morpholino antisense oligonucleotides are potent inhibitors of HCV IRES translation in a preclinical mouse model; morpholinos have potential as molecular therapeutics for treating HCV and other viral infections. The in vivo model described is a broadly applicable, straightforward, and rapid readout for inhibitor efficacy. As such, it will greatly facilitate the development of novel therapeutic strategies for viral hepatitis. Notably, the level of antisense inhibition observed in this in vivo model is similar to the maximal inhibition we have obtained previously with RNA interference in mice.

Abstract

Heme oxygenase (HO) is the rate-limiting step in the heme degradation pathway and is a potential target for the control, or prevention, of pathologic jaundice in neonates. Metalloporphyrins (Mps), a diverse set of synthetic derivatives of heme, can competitively inhibit the HO enzymes. However, certain Mps are phototoxic and some increase transcription of HO-1, the inducible HO isozyme. Therefore, effective development of this class of compounds as therapeutics for treating pathologic jaundice will require rapid and integrated biological screens to identify the most efficacious and safe Mps. To study the safety of these compounds, we assessed their cytotoxic effects and measured luciferase activity by bioluminescent imaging (BLI) as an index of HO-1 transcription, first in live cell cultures and then in living transgenic reporter mice. A total of 12 Mps were first evaluated in the correlative cell culture assay. Based on results from this study, 2 Mps, zinc protoporphyrin (ZnPP) and zinc bis glycol porphyrin (ZnBG), were selected for further studies in the live animal model. In vitro BLI showed ZnPP to be a strong inducer of HO-1 transcription in comparison to ZnBG, which showed minimal induction. Cytotoxicity studies revealed that ZnPP was phototoxic, whereas ZnBG had no effect on cell viability. In vivo BLI showed that both ZnPP and ZnBG had minimal effects on the levels of HO-1 transcription in the animals. Furthermore, serum enzyme assays indicated that neither caused detectable liver toxicity. These findings, and especially those with ZnBG, support the use of selected Mps as therapies for pathologic jaundice. Coupling the high throughput advantage of cell culture with the capability of imaging for whole-body temporal analyses could accelerate and refine the preclinical phases of drug development. Thus, this study serves as a model for understanding the effects of specific compounds in relation to defined targets using an integrated approach.

Abstract

Human cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of numerous xenobiotics in the human liver. We have examined the activation of the human CYP3A4 promoter in mouse liver by using in vivo bioluminescent imaging (BLI). Transcription of the CYP3A4 promoter occurs as a result of a ligand binding to a nuclear orphan receptor, pregnane X receptor (PXR), followed by dimerization with another nuclear receptor, retinoid X receptor (RXR). Since this heterodimer then binds to xenobiotic response elements to activate transcription of CYP3A4, we examined a 13kb promoter region of CYP3A4 for responsiveness to dexamethasone and rifampicin. A reporter vector CYP3A4-luc was constructed consisting of the CYP3A4 promoter driving the firefly luciferase gene. This DNA was injected into the tail veins of mice, and reporter gene expression was monitored in the liver region using BLI. Treatment of transfected mice with dexamethasone resulted in a 188-fold induction of luciferase, whereas treatment with rifampicin resulted in a 68-fold induction. Co-injection with a human PXR expression vector resulted in a dramatic increase in rifampicin-induced activity and a smaller increase of dexamethasone-induced activity. Co-injection of an antisense murine PXR construct with the CYP3A4-luc reduced both the dexamethasone- and rifampicin-induced responses, thus demonstrating that the murine PXR receptor can participate in the regulation of the human CYP3A4 promoter in mice. The approach described here will be of general use in studying the regulation of nuclear receptors in vivo.

Abstract

We report on the use of optical techniques to monitor and treat Pseudomonas aeruginosa wound infections in mice. Bioluminescent bacteria transduced with a plasmid containing a bacterial lux gene operon allow the infection in excisional mouse wounds to be imaged by use of a sensitive charge-coupled device camera. Photodynamic therapy (PDT) targeted bacteria, by use of a polycationic photosensitizer conjugate, which is designed to penetrate the gram-negative cell wall and was topically applied to the wound and was followed by red-light illumination. There was a rapid light dose-dependent loss of luminescence, as measured by image analysis, in the wounds treated with conjugate and light, a loss that was not seen in untreated wounds, wounds treated with light alone, or wounds treated with conjugate alone. P. aeruginosa was invasive in our mouse model, and all 3 groups of control mice died within 5 days; in contrast, 90% of PDT-treated mice survived. PDT-treated wounds healed significantly faster than did silver nitrate-treated wounds, and this was not due to either inhibition of healing by silver nitrate or stimulation of healing by PDT.

Abstract

Effective development of therapeutics that target the molecular basis of disease is dependent on testing new therapeutic moieties and delivery strategies in animal models of human disease. Accelerating the analyses of these models and improving their predictive value through whole animal imaging methods, which provide data in real time and are sensitive to the subtle changes, are crucial for rapid advancement of these approaches. Modalities based on optics are rapid, sensitive, and accessible methods for in vivo analyses with relatively low instrumentation costs. In vivo bioluminescent imaging (BLI) is one of these optically based imaging methods that enable rapid in vivo analyses of a variety of cellular and molecular events with extreme sensitivity. BLi is based on the use of light-emitting enzymes as internal biological light sources that can be detected externally as biological indicators. BLI has been used to test spatio-temporal expression patterns of both target and therapeutic genes in living laboratory animals where the contextual influences of whole biological systems are preserved. BLI has also been used to analyze gene delivery, immune cell therapies, and the in vivo efficacy of inhibitory RNAs. New tools for BLI are being developed that will offer greater flexibility in detection and analyses. BLI can be used to accelerate the evaluation of experimental therapeutic strategies and whole body imaging offers the opportunity of revealing the effects of novel approaches on key steps in disease processes.

Abstract

We describe a novel confocal microscope that uses separate low-numerical-aperture objectives with the illumination and collection axes crossed at angle theta from the midline. This architecture collects images in scattering media with high transverse and axial resolution, long working distance, large field of view, and reduced noise from scattered light. We measured transverse and axial (FWHM) resolution of 1.3 and 2.1 microm, respectively, in free space, and confirm subcellular resolution in excised esophageal mucosa. The optics may be scaled to millimeter dimensions and fiber coupled for collection of high-resolution images in vivo.

Abstract

Animal models are important tools to investigate the pathogenesis and develop treatment strategies for bone metastases in humans. However, there are few spontaneous models of bone metastasis despite the fact that rodents (rats and mice) and other animals (dogs and cats) often spontaneously develop cancer. Therefore, most experimental models of bone metastasis in rodents require injection or implantation of neoplastic cells into orthotopic locations, bones, or the left ventricle of the heart.The current study reviews the natural incidence and clinical manifestation of bone metastases of mammary and prostate carcinoma in animals, as well as the experimental models developed in mice using animal and human-derived neoplasms.Rats, mice, dogs, and cats often develop spontaneous mammary carcinoma, but bone metastases are rare. Intact and neutered dogs develop prostate carcinoma that is usually androgen independent and may be associated with regional bone invasion or distant bone metastasis. Normal dog prostate tissue induces new bone formation in vivo and can serve as a model of osteoblastic metastasis without concurrent bone destruction. Experimental models of osteolytic, osteoblastic, and mixed osteolytic/osteoblastic bone metastases include syngeneic rodent neoplasms or human xenografts implanted at orthotopic sites (e.g., breast or prostate glands) in immunodeficient mice, injection of cancer cells into the left ventricle of the heart, or direct injection into bones. New transgenic mouse models of cancer have a low incidence of spontaneous bone metastasis, but cell lines derived from these tumors can be selected in vivo for increased incidence of bone metastasis. It is essential to validate and correctly interpret the lesions in models of bone metastasis to accurately correlate the data from animal models to human disease. Animal models have provided support for the "seed and soil" hypothesis of bone metastasis. However, the roles of vascular patterns in the metaphyses of long bones and rapid bone turnover in young animals in the pathogenesis of metastasis in experimental models are uncertain. Improvements in the imaging of experimental animals in vivo using fluorescent markers or light emitted from luciferase have led to increased sensitivity of detection and more accurate quantification of bone metastases. For example, imaging of human prostate carcinoma PC-3M cells transfected with luciferase, following injection into the left ventricle, has demonstrated that there is rapid localization of tumor cells to bones and other organs, such as the kidneys and lungs.Animal models of metastasis have supported drug development and have been useful for identification of metastasis suppressor and promoter genes as novel targets for the development of novel therapies. Further refinement of these models will involve spatiotemporal analysis of the metastatic process by imaging and use of image data to stage disease and guide tissue sampling for gene expression profiling via gene array technology. In the future, integrated analyses of these models will be needed to understand the complexities of this important disease process.

Abstract

Cancer therapeutics have achieved success in the treatment of a variety of malignancies, however, relapse of disease from small numbers of persistent tumor cells remains a major obstacle. Advancement of treatment regimens that effectively control minimal residual disease and prevent relapse would be greatly accelerated if sensitive and noninvasive assays were used to quantitatively assess tumor burden in animal models of minimal residual disease that are predictive of the human response. In vivo bioluminescence imaging (BLI) is an assay for the detection of small numbers of cells noninvasively and enables the quantification of tumor growth within internal organs. Fusion genes that encode bioluminescent and fluorescent reporter proteins effectively couple the powerful in vivo capabilities of BLI with the subset-discriminating capabilities of fluorescence-activated cell sorting. We labeled 2 murine lymphoma cell lines with dual function reporter genes and monitored radiation and chemotherapy as well as immune-based strategies that employ the tumorcidal activity of ex vivo-expanded CD8(+) natural killer (NK)-T cells. Using BLI we were able to visualize the entire course of malignant disease including engraftment, expansion, metastasis, response to therapy, and unique patterns of relapse. We also labeled the effector NK-T cells and monitored their homing to the sites of tumor growth followed by tumor eradication. These studies reveal the efficacy of immune cell therapies and the tempo of NK-T cell trafficking in vivo. The complex cellular processes in bone marrow transplantation and antitumor immunotherapy, previously inaccessible to investigation, can now be revealed in real time in living animals.

Abstract

Myelotoxic treatments for oncologic diseases are often complicated by neutropenia, which renders patients susceptible to potentially lethal infections. In these studies of murine hematopoietic stem cell transplantation (HSCT), cotransplantation of lineage-restricted progenitors known as common myeloid progenitors (CMP) and granulocyte-monocyte progenitors (GMP) protects against death following otherwise lethal challenge with either of 2 pathogens associated with neutropenia: Aspergillus fumigatus and Pseudomonas aeruginosa. Cotransplantation of CMP/GMP resulted in a significant and rapid increase in the absolute number of myeloid cells in the spleen, most of which were derived from the donor CMP/GMP. Despite persistent peripheral neutropenia, improved survival correlated with the measurable appearance of progenitor-derived myeloid cells in the spleen. A marked reduction or elimination of tissue pathogen load was confirmed by culture and correlated with survival. Localization of infection by P aeruginosa and extent of disease was also assessed by in vivo bioluminescent imaging using a strain of P aeruginosa engineered to constitutively express a bacterial luciferase. Imaging confirmed that transplantation with a graft containing hematopoietic stem cells and CMP/GMP reduced the bacterial load as early as 18 hours after infection. These results demonstrate that enhanced reconstitution of a tissue myeloid pool offers protection against lethal challenge with serious fungal and bacterial pathogens.

Abstract

Rheumatoid arthritis (RA) is an autoimmune arthritis, for which treatment options remain limited. This study investigated the potential role of adoptive cellular gene therapy as a novel means for treating the RA animal model collagen-induced arthritis (CIA). Adoptive transfer of antigen-specific T-cell hybridomas retrovirally transduced to express IL-4 1 day before booster immunization significantly reduced the number of inflamed joints. Cell transfer after clinical onset of disease had no therapeutic effect. Bioluminescence imaging showed that the hybridomas migrated to the inflamed joints, thus delivering the regulatory protein locally at the site of inflammation. The homing was, at least in part, due to chemotaxis in response to proinflammatory chemokines that are expressed in inflamed joints. There were no significant changes in the cytokine milieu of the draining lymph nodes, nor in the systemic levels of anti-collagen antibodies in treated mice. We conclude that the beneficial clinical effects observed in our model were most likely based on the local action(s) of IL-4 in the inflamed joints and that the local delivery (and effects) of regulatory cytokines, like IL-4, constitutes a novel and effective method of preventing organ-specific autoimmune disease and of minimizing systemic adverse effects.

Abstract

Malignant disease is the final manifestation of complex molecular and cellular events leading to uncontrolled cellular proliferation and eventually tissue destruction and metastases. While the in vitro examination of cultured tumour cells permits the molecular dissection of early pathways in tumorigenesis on cellular and subcellular levels, only interrogation of these processes within the complexity of organ systems of the living animal can reveal the full range of pathophysiological changes that occur in neoplastic disease. Such analyses require technologies that facilitate the study of biological processes in vivo, and several approaches have been developed over the last few years. These strategies, in the nascent field of in vivo molecular and cellular imaging, combine molecular biology with imaging modalities as a means to real-time acquisition of functional information about disease processes in living systems. In this review, we will summarise recent developments in in vivo bioluminescence imaging (BLI) and discuss the potential of this imaging strategy for the future of cancer research.

Abstract

HER2 is an attractive immunotherapeutic target for neoplastic disease because this cell surface molecule is overexpressed on a large fraction of malignant tumor cells. To directly assess therapeutic responses to targeted therapy by noninvasive in vivo imaging in small animals, human HER2-expressing ovarian carcinoma cells were genetically modified with a firefly luciferase gene, and light emission was used for visualization of tumor growth and response to therapy. This imaging approach was able to demonstrate in real-time tumor regression in a HER2 xenograft mouse model by adoptive transfer of in vitro induced and expanded cytotoxic CD8+ natural killer T (NKT) cells retargeted with a humanized bispecific antibody F(ab')(2)HER2xCD3. Immunotherapy with effector cells alone or a humanized monoclonal antibody anti-p185(HER2) (4D5-8) resulted in significant but slower reduction in tumor burden. Long-term survival of tumor xenografts correlated inversely with visible residual tumor burden. In vitro, F(ab')(2)HER2xCD3 substantially augmented cytotoxic activity of CD8+ NKT cells. By flow-sorting, CD8+ NKT cells coexpressing CD56 were found to have the highest redirected killing ability. Treatment with concanamycin A or EGTA abrogated CD8+ NKT cytotoxicity indicating that perforin is a major pathway of tumor cell lysis. In contrast, when CD8+ NKT cell were cross-linked with F(ab')(2)HER2xCD3 neither the immunosuppressants cyclosporine A and FK506, nor the increase of intracellular cyclic AMP by dibutyryl cyclic AMP were able to inhibit cytotoxicity demonstrating that signaling via the CD3 antigen changes the biological activity of non-MHC-restricted effector cells. These studies have demonstrated that CD8+ NKT cells can be successfully redirected to tumor cells using bispecific antibodies and offer a promising strategy for adoptive immunotherapy of neoplastic diseases.

It's not just about anatomy: In vivo bioluminescence imaging as an eyepiece into biologyJOURNAL OF MAGNETIC RESONANCE IMAGINGContag, C. H., Ross, B. D.2002; 16 (4): 378-387

Abstract

Among the newly described tools that enable analyses of cellular and molecular events in living animals, in vivo bioluminescence imaging (BLI) offers important opportunities for investigating a wide variety of disease processes. BLI utilizes luciferase as an internal biological light source that can be genetically programmed to noninvasively "report" the presence or activation of specific biological events. Applications of BLI have included the use of luciferase to demonstrate expression of cell- and tissue-specific promoters, label cell populations, guide detection by other imaging modalities, and detect protein-protein interaction. These applications of BLI technology have allowed quantitative measurements of tumor burden and treatment response, immune cell trafficking, and detection of gene transfer. Spatiotemporal information can be rapidly obtained in the context of whole biological systems in vivo, which can accelerate the development of experimental therapeutic strategies. This paper provides a review of the biological applications in which in vivo BLI has been utilized to nondestructively monitor biological processes in intact small animal models, and highlights some of the advancements that will increase the versatility of BLI as a molecular imaging tool.

Abstract

Heme oxygenase (HO), a key catabolic enzyme in the conversion of heme to bilirubin, is an ideal target for reducing bilirubin production and preventing pathological jaundice in newborn infants. Metalloporphyrins (Mps) have been well characterized as competitive inhibitors of HO and have been evaluated as potential chemopreventive agents for neonatal jaundice. However, in addition to reducing HO activity, many Mps have been shown to increase HO-1 transcription, which would likely reduce their potential therapeutic utility. The differential effects of Mps on the transcription of HO-1 were therefore evaluated in living transgenic (Tg) reporter mice. Of the compounds evaluated, we observed that zinc bis-glycol porphyrin (ZnBG), a potent inhibitor of HO enzyme activity, did not alter HO-1 transcription patterns in Tg mice. Whole body images of HO-1 transcription patterns did, however; reveal increases in HO-1 transcription in Tg mice after treatment with other Mps, heme and cadmium chloride (CdCl(2)). Intravenous injections of CdCl(2) resulted in expression patterns that differed in tempo and location from those observed in Tg mice treated with intraperitoneal injections. Spatiotemporal analyses of transcriptional regulation in living animals accelerated the assessment of an adverse effect of Mps by revealing different patterns of HO-1 transcription. Among the known inhibitors of HO enzyme activity that were evaluated in this study, ZnBG did not significantly affect HO-1 transcription and therefore may be well suited for the prevention of neonatal jaundice.

Abstract

Autoimmune disorders represent inappropriate immune responses directed at self-tissue. Because CD4+ T cells are important mediators in the pathogenesis of autoimmune disease, they are ideal candidates for cell-based gene therapy. Using retrovirally-transduced cells and luciferase bioluminescence, we have demonstrated that primary T cells and hybridomas, rapidly and preferentially home to the sites of inflammation in organ-specific autoimmune disease. These cells, transduced with retroviral vectors to drive expression of various 'regulatory proteins', such as IL-4, IL-10 and IL-12p40, deliver these immunoregulatory proteins to the inflamed lesions, providing therapy for experimental models of autoimmune disease such as EAE, CIA and NOD mice. This technique was originally developed in our lab in the murine model of multiple sclerosis, EAE, where T cell hybridomas reactive with myelin basic protein (MBP) were transduced to express and used to deliver the modulatory cytokine, IL-4. Recently we have observed that the cytokine receptor antagonist, IL-12p40 transduced anti-myelin basic protein (MBP) TCR-transgenic T cells (but not CII-reactive T cells) were effective in preventing EAE whereas the CII-reactive, but not MBP-reactive T cells, transduced to express IL-12p40, would treat CIA.

Abstract

Bone morphogenetic protein 4 (Bmp4) plays a significant role in development. Using transgenic approaches, we studied the mechanisms controlling Bmp4 expression during primordial and mature tissue development, as well as in epithelium- and mesenchyme-derived components with hair follicles as a model. In this report, we demonstrated that the promoter region between the -0.26 and the -1.14 kb, highly conserved between human and mouse, controls Bmp4 expression in the epithelium-derived tissues but not in mesenchyme-derived tissues of hair follicles, suggesting that control modules for Bmp4 expression in epithelium-derived tissues and mesenchyme originated tissues are in separate regions. Using live embryos and mice, we demonstrated the temporal and spatial activities of these modules. We also demonstrated that control regions for Bmp4 expression in primordial and differentiated hair follicle tissues are separated. Therefore we present a model to explain a mechanism controlling expression of the Bmp4 gene in different tissue types, as well as different development stages as related to hair development.

Abstract

Lactase gene transcription is spatially restricted to the proximal and middle small intestine of the developing mouse. To identify regions of the lactase gene involved in mediating the spatiotemporal expression pattern, transgenic mice harboring 0.8-, 1.3-, and 2.0-kb fragments of the 5'-flanking region cloned upstream of a firefly-luciferase reporter were generated. Transgene expression was assessed noninvasively in living mice using a sensitive low light imaging system. Two independent, 1.3- and 2.0-kb, lactase promoter-reporter transgenic lines expressed appropriate high levels of luciferase activity in the small intestine (300-3,000 relative light units/microg) with maximal expression in the middle segments. Post-weaned 30-day transgenic offspring also demonstrated an appropriate 4-fold maturational decline in luciferase expression in the small intestine. The pattern of the 2.0-kb promoter transgene mRNA abundance most closely mimicked that of the endogenous lactase gene with respect to spatiotemporal restriction. In contrast, a 0.8-kb promoter-reporter construct expressed low level luciferase activity (<25 relative light units/microg) in multiple organs and throughout the gastrointestinal tract in transgenic mice. Thus, a distinct 5'-region of the lactase promoter directs intestine-specific expression in the small intestine of transgenic mice, and regulatory sequences have been localized to a 1.2-kb region upstream of the lactase transcription start site. In addition, we have demonstrated that in vivo bioluminescence imaging can be utilized for assessment of intestinal expression patterns of a luciferase reporter gene driven by lactase promoter regions in transgenic mice.

Abstract

Cell migration is a key aspect of the development of the immune system and mediating an immune response. There is extensive and continual redistribution of cells to different anatomic sites throughout the body. These trafficking patterns control immune function, tissue regeneration, and host responses to insult. The ability to monitor the fate and function of cells, therefore, is imperative to both understanding the role of specific cells in disease processes and to devising rational therapeutic strategies. Determining the fate of immune cells and understanding the functional changes associated with migration and proliferation require effective means of obtaining in vivo measurements in the context of intact organ systems. A variety of imaging methods are available to provide structural information, such as X-ray CT and MRI, but only recently new tools have been developed that reveal cellular and molecular changes as they occur within living animals. We have pioneered one of these techniques that is based on the observations that light passes through mammalian tissues, and that luciferases can serve as internal biological sources of light in the living body. This method, called in vivo bioluminescence imaging, is a rapid and noninvasive functional imaging method that employs light-emitting reporters and external photon detection to follow biological processes in living animals in real time. This imaging strategy enables the studies of trafficking patterns for a variety of cell types in live animal models of human biology and disease. Using this approach we have elucidated the spatiotemporal trafficking patterns of lymphocytes within the body. In models of autoimmune disease we have used the migration of "pathogenic" immune cells to diseased tissues as a means to locally deliver and express therapeutic proteins. Similarly, we have determined the tempo of NK-T cell migration to neoplastic lesions and measured their life span in vivo. Using bioluminescence imaging individual groups of animals can be followed over time significantly reducing the number of animals per experiment, and improving the statistical significance of a study since changes in a given population can be studied over time. Such rapid assays that reveal cell fates in vivo will increase our basic understanding of the molecular signals that control these migratory pathways and will substantially speed up the development and evaluation of therapies.

Abstract

The worldwide rise in antibiotic resistance necessitates the development of novel antimicrobial strategies. In this study we report on the first use of a photochemical approach to destroy bacteria infecting a wound in an animal model. Following topical application, a targeted polycationic photosensitizer conjugate between poly-L-lysine and chlorin(e6) penetrated the gram (-) outer bacterial membrane, and subsequent activation with 660 nm laser light rapidly killed Escherichia coli infecting excisional wounds in mice. To facilitate real-time monitoring of infection, we used bacteria that expressed the lux operon from Photorhabdus luminescens; these cells emitted a bioluminescent signal that allowed the infection to be rapidly quantified, using a low-light imaging system. There was a light-dose dependent loss of luminescence in the wound treated with conjugate and light, not seen in untreated wounds. Treated wounds healed as well as control wounds, showing that the photodynamic treatment did not damage the host tissue. Our study points to the possible use of this methodology in the rapid control of wounds and other localized infections.

Abstract

To advance our understanding of biological processes as they occur in living animals, imaging strategies have been developed and refined that reveal cellular and molecular features of biology and disease in real time. One rapid and accessible technology for in vivo analysis employs internal biological sources of light emitted from luminescent enzymes, luciferases, to label genes and cells. Combining this reporter system with the new generation of charge coupled device (CCD) cameras that detect the light transmitted through the animal's tissues has opened the door to sensitive in vivo measurements of mammalian gene expression in living animals. Here, we review the development and application of this imaging strategy, in vivo bioluminescence imaging (BLI), together with in vivo fluorescence imaging methods, which has enabled the real-time study of immune cell trafficking, of various genetic regulatory elements in transgenic mice, and of in vivo gene transfer. BLI has been combined with fluorescence methods that together offer access to in vivo measurements that were not previously available. Such studies will greatly facilitate the functional analysis of a wide range of genes for their roles in health and disease.

Abstract

Gene fusions composed of specific promoters and bioluminescent reporter genes can be used to assess gene expression patterns using whole-body imaging in living animal models. A transgenic mouse model was developed using the regulatory elements of the heme oxygenase promoter to drive luciferase as the reporter gene. In these transgenic mice, heme oxygenase (HO)-1 expression was apparent in neuronal tissues of neonates but not adults as measured by whole-body imaging, and in adults transcription of the reporter gene was inducible by known inducers of HO-1 transcription. Whole-body imaging of luciferase activity was then used to evaluate the effects of metalloporphyrins (Mps) on the transcription of the reporter gene. Some of the Mps, which are potent inhibitors of HO activity, did not activate the reporter gene above background. These Mps are ideally suited as chemotherapeutics that may target bilirubin production rates by inhibiting HO activity, but not result in a net increase in output from the HO gene. In contrast, known inducers of HO transcription did increase luciferase activity as did some of the other Mps that have been examined. Using whole-body in vivo transcriptional assays may facilitate rapid screening of potential therapeutic compounds for both desired and untoward effects.

Abstract

Lymphocytes are highly mobile cells that travel throughout the body in response to a tremendous variety of stimuli. Revealing lymphocyte trafficking patterns in vivo is necessary for a complete understanding of immune function, as well as cell-cell and cell-tissue interactions in immune development and in response to insult. Although the location of cell populations in various tissues at any given point in time may be revealed by techniques such as flow cytometry and immunofluorescence, these methods are not readily amenable to the assessment of dynamic cell migration patterns in vivo. In the past 5 years, technologies for imaging molecular and cellular changes in living animals have advanced to a point where it is possible to reveal the migratory paths of these vitally important cells. Here, we review one advancement in cellular imaging, in vivo bioluminescence imaging, which addresses the problem of lymphocyte tracking. This imaging strategy has the potential to elucidate the temporal patterns of immune responses and the spatial distribution of lymphocytes within the body.

Abstract

Autoantigen-specific T cells have tissue-specific homing properties, suggesting that these cells may be ideal vehicles for the local delivery of immunoregulatory molecules. We tested this hypothesis by using type II collagen-specific (CII-specific) CD4(+) T hybridomas or primary CD4(+) T cells after gene transfer, as vehicles to deliver an immunoregulatory protein for the treatment of collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). CII-specific T cells or hybridomas were transduced using retroviral vectors to constitutively express the IL-12 antagonist, IL-12 p40. Transfer of engineered CD4(+) T cells after immunization significantly inhibited the development of CIA, while cells transduced with vector control had no effect. The beneficial effect on CIA of IL-12 p40-transduced T cells required TCR specificity against CII, since transfer of T cells specific for another antigen producing equivalent amounts of IL-12 p40 had no effect. In vivo cell detection using bioluminescent labels and RT-PCR showed that transferred CII-reactive T-cell hybridomas accumulated in inflamed joints in mice with CIA. These results indicate that the local delivery of IL-12 p40 by T cells inhibited CIA by suppressing autoimmune responses at the site of inflammation. Modifying antigen-specific T cells by retroviral transduction for local expression of immunoregulatory proteins thus offers a promising strategy for treating RA.

Abstract

Neonatal hyperbilirubinemia is a normal postnatal phenomenon resulting from a transitional imbalance between the production and elimination of bilirubin in the neonate. Bilirubin has been shown to be not only a potent antioxidant, but also toxic at excessive concentrations. As a result, the biology of bilirubin, its production, regulation, and measurements have been the focus of extensive studies. Bilirubin, carbon monoxide, and iron are derived from the degradation of heme, a ubiquitous two-step pathway catalyzed by the enzyme, heme oxygenase. It has been shown that these metabolically active products from the heme catabolic pathway may, in turn, influence many other biologic processes. This report provides a brief overview of these interrelationships in the hope that it may provide insight into the central role this pathway plays in the existence of most organisms.

Abstract

Recombinant adeno-associated viruses (rAAV) are promising gene transfer vectors that produce long-term expression without toxicity. To investigate future approaches for in utero gene delivery, the efficacy and safety of prenatal administration of rAAV were determined. Using luciferase as a reporter, expression was assessed by whole-body imaging and by analysis of luciferase activity in tissue extracts, at the time of birth and monthly thereafter. Transgene expression was detected in all injected animals. Highest levels of luciferase activity were detected at birth in the peritoneum and liver, while the heart, brain, and lung demonstrated low-level expression. In vivo luciferase imaging revealed persistent peritoneal expression for 18 months after in utero injection and provided a sensitive whole-body assay, useful in identifying tissues for subsequent analyses. There was no detectable hepatocellular injury. Antibodies that reacted with either luciferase or rAAV were not found. AAV sequences were not detected in germ-line tissues of injected animals or in tissues of their progeny. In utero AAV-mediated gene transfer in this animal model demonstrates that novel therapeutic vectors and strategies can be rapidly tested in vivo and that rAAV may be developed to ameliorate genetic diseases with perinatal morbidity and mortality.

Abstract

The use of transgenic animals in biomedical research is increasing rapidly and may be the best means of determining gene function. Generating transgenic animals typically requires time-consuming screening processes, and gene function is assessed by an array of difficult phenotypic and biochemical assays performed ex vivo. To address the unmet need in transgenic research for functional assays performed with ease in living animals, we demonstrate here that in vivo detection of luciferase enzyme as a transcriptional reporter facilitates rapid screening for both the presence and function of transgenes in intact living mice. Using this approach we identified three bioluminescent transgenic founders where the transgene consisted of the heme oxygenase promoter fused to the modified coding sequence of the luciferase gene. These founders were identified from 183 pups and confirmed by PCR analysis. Identification of HO-1-luc homozygotes from back- crossed F2 littermates was then accelerated by in vivo imaging. In another transgenic mouse line, where the transgene was comprised of the bone morphogenic-4 (BMP4) promoter fused to the modified luciferase gene, we were able to identify transgenic animals and in each line we were able to visualize patterns of expression in living animals over time. The light production from these transgenic mice indicated that the desired DNA fragment was functional and different expression profiles apparent at different ages and after gene induction.

Abstract

Maternal and cord samples from HIV-seropositive women and their infants in Zimbabwe, where subtype C is the predominant strain of HIV, were analyzed to determine the frequency of detection of HIV RNA and DNA. HIV RNA was detected in 90% of maternal and in 38% of cord plasma at levels at least 25% of maternal plasma. Heteroduplex mobility assays and sequencing of virus envelope (C2-V5) demonstrated closely related, but unique, subtype C viruses in maternal and cord RNA, and a significantly greater frequency of cord viremia among women with homogenous, compared with heterogeneous viral envelope RNA. Quantification of RNA, measures of envelope viral diversity, and phylogenetic analysis of maternal and cord plasma RNA provide evidence for the frequent exposure and potential transmission of HIV from mother to infant before birth.

Abstract

Current assessment of orthotopic tumor models in animals utilizes survival as the primary therapeutic end point. In vivo bioluminescence imaging (BLI) is a sensitive imaging modality that is rapid and accessible, and may comprise an ideal tool for evaluating antineoplastic therapies. Using human tumor cell lines constitutively expressing luciferase, the kinetics of tumor growth and response to therapy have been assessed in intraperitoneal, and subcutaneous, and intravascular cancer models. However, use of this approach for evaluating orthotopic tumor models has not been demonstrated. In this report, the ability of BLI to noninvasively quantitate the growth and therapeutic-induced cell kill of orthotopic rat brain tumors derived from 9L gliosarcoma cells genetically engineered to stably express firefly luciferase (9LLuc) was investigated. Intracerebral tumor burden was monitored over time by quantitation of photon emission and tumor volume using a cryogenically cooled CCD camera and magnetic resonance imaging (MRI), respectively. There was excellent correlation (r=0.91) between detected photons and tumor volume. A quantitative comparison of tumor cell kill determined from serial MRI volume measurements and BLI photon counts following 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment revealed that both imaging modalities yielded statistically similar cell kill values (P=.951). These results provide direct validation of BLI imaging as a powerful and quantitative tool for the assessment of antineoplastic therapies in living animals.

Abstract

Our previous studies have indicated that HIV transmission from infected mothers to infants occurs with viruses showing rapid kinetics of replication, and either resistance to maternal neutralizing antibodies or sensitivity to enhancing antibodies. The genotypic patterns that result in these and other phenotypic viral characteristics may provide clues to the selection pressures exerted during this mode of transmission. For this reason, DNA sequences of the envelope gene (env) were determined for viral isolates obtained from seropositive women who were mothers of either infected or uninfected infants. Sequences of viruses isolated early in life from the infected newborns were also determined, such that diversity both within isolates and between maternal and infant isolates could be assessed. Among isolates obtained from mothers of uninfected infants, the V3 region of env demonstrated a higher degree of heterogeneity than those from mothers of infected infants. Similar to the viruses obtained from the mothers of infected infants, the infant-derived viral sequences were relatively homogeneous. Finally, the reactivity of maternal plasma with infant-derived HIV isolates, whether via neutralizing or enhancing antibodies, appeared to predict the distribution of viral sequences in the infant isolates. These data suggest that selective pressure on HIV-1 during transmission or growth in the infected infant may be mediated by biologic and/or immunologic processes.

Abstract

HIV-1 envelope sequence patterns have implications for virus cell tropism and for the development of an effective vaccine. To identify the sequence characteristics of recently transmitted HIV-1 isolates in southern Africa, we sequenced the V3-V5 envelope regions of 24 male seroconverters in Harare, Zimbabwe. Each of the sequences clustered with previously reported subtype C isolates and there was a mean 17% intersequence pairwise genetic distance between the Zimbabwean isolates. Three isolates were syncytium inducing (SI). One of the SI isolates had an unusual GIGK crown and a deletion at codon 23; one had the codon 23 deletion alone; and one had a high net positive charge in the V3 loop. The extensive genetic diversity within the envelope of subtype C HIV-1 isolates must be considered in vaccine development. Further analysis of subtype C SI isolates and site-directed mutagenesis experiments are required to determine the molecular basis of SI activity in global HIV-1 isolates.

Abstract

Somatic cell gene transfer is a potentially useful strategy to alter lung function. However, achieving efficient transfer to the alveolar epithelium, especially in smaller animals, has not been demonstrated. In this study, the rat heme oxygenase-1 (HO-1) gene was delivered to the lungs of neonatal mice via transpulmonary injection. A bidirectional promoter construct coexpressing both HO-1 and a luciferase reporter gene was used so that in vivo gene expression patterns could be monitored in real time. HO-1 expression levels were also modulated with doxycycline and assessed in vivo with bioluminescent light transmitted through the tissues from the coregulated luciferase reporter. As a model of oxidative stress and HO-1-mediated protection, groups of animals were exposed to hyperoxia. After gene transfer, elevated levels of HO-1 were detected predominantly in alveolar type II cells by immunocytochemistry. With overexpression of HO-1, increased oxidative injury was observed. Furthermore, this model demonstrated a cell-specific effect of lung HO-1 overexpression in oxidative stress. Specific control of expression for therapeutic genes is possible in vivo. The transpulmonary approach may prove useful in targeting gene expression to cells of the alveolar epithelium or to circumscribed areas of the lung.

Abstract

Revealing the cellular and molecular changes associated with cancer, as they occur in intact living animal models of human neoplastic disease, holds tremendous potential for understanding disease mechanisms and elucidating effective therapies. Since light is transmitted through mammalian tissues, at a low level, optical signatures conferred on tumor cells by expression of reporter genes encoding bioluminescent and fluorescent proteins can be detected externally using sensitive photon detection systems. Expression of reporter genes, such as the bioluminescent enzyme firefly luciferase (Luc) or variants of green fluorescent protein (GFP) in transformed cells, can effectively be used to reveal molecular and cellular features of neoplasia in vivo. Tumor cell growth and regression in response to various therapies have been evaluated non-invasively in living experimental animals using these reporter genes. Detection of Luc-labeled cells in vivo was extremely sensitive with signals over background from as few as 1000 human tumor cells distributed throughout the peritoneal cavity of a mouse with linear relationships between cell number and signal intensity over five logs. GFP offers the strength of high-resolution ex vivo analyses following in vivo localization of the tumor. The dynamic range of Luc detection allows the full disease course to be monitored since disease progression from small numbers of cells to extensive disease can be assessed. As such, therapies that target minimal disease as well as those designed for late stage disease can be readily evaluated in animal models. Real time spatiotemporal analyses of tumor cell growth can reveal the dynamics of neoplastic disease, and facilitate rapid optimization of effective treatment regimens. Thus, these methods improve the predictability of animal models of human disease as study groups can be followed over time, and can accelerate the development of therapeutic strategies.

Abstract

Even though the heme degradation pathway consists of only two reactions, it and its major enzyme (i.e. HO), nonetheless, impact other processes not only through the removal of excess heme, but also through the production of several metabolically active compounds. Thus CO and biliverdin along with reactive iron, Fe2, are the primordial products of this ancient, highly conserved reaction. That every component of the heme catabolic pathway is directly or indirectly related to other reactions involving oxygen or light is, perhaps, no accident of nature. That a fundamentally destructive event can be linked with a multiplicity of synthetic events and various biological effects, depending on the timing and location of the HO activity, is testament to the economy and the ultimate beauty of nature. Furthermore, the interaction of the heme catabolic pathway with that of the NOS system may lead to even more exciting avenues of research. It may be shown that the integrity of the heme catabolic pathway, which is ever present and plays a role in every tissue, is central to the existence of most complex organisms.

Abstract

Evaluation of potential antineoplastic therapies would be enhanced by noninvasive detection of tumor cells in living animals. Because light is transmitted through mammalian tissues, it was possible to use bioluminescence to monitor (both externally and quantitatively) growth and regression of labeled human cervical carcinoma (HeLa) cells engrafted into immunodeficient mice. The efficacy of both chemotherapy and immunotherapeutic treatment with ex vivo expanded human T cell-derived effector cells was evaluated. In the absence of therapy, animals showed progressive increases in signal intensity over time. Animals treated with cisplatin had marked reductions in tumor signal; 5'-fluorouracil was less effective, and cyclophosphamide was ineffective. Immunotherapy dramatically reduced signals at high effector-to-target cell ratios, and significant decreases were observed with lower ratios. This model system allowed sensitive, quantitative, real-time spatiotemporal analyses of the dynamics of neoplastic cell growth and facilitated rapid optimization of effective treatment regimens.

Abstract

Revealing the mechanisms of neoplastic disease and enhancing our ability to intervene in these processes requires an increased understanding of cellular and molecular changes as they occur in intact living animal models. We have begun to address these needs by developing a method of labeling tumor cells through constitutive expression of an optical reporter gene, and noninvasively monitoring cellular proliferation in vivo using a sensitive photon detection system. A stable line of HeLa cells that expressed a modified firefly luciferase gene was generated, and proliferation of these cells in irradiated severe combined immunodeficiency (SCID) mice was monitored. Tumor cells were introduced into animals via subcutaneous, intraperitoneal and intravenous inoculation and whole body images, that revealed tumor location and growth kinetics, were obtained. The number of photons that were emitted from the labeled tumor cells and transmitted through murine tissues was sufficient to detect 1x10(3) cells in the peritoneal cavity, 1x10(4) cells at subcutaneous sites and 1x10(6) circulating cells immediately following injection. The kinetics of cell proliferation, as measured by photon emission, was exponential in the peritoneal cavity and at subcutaneous sites. Intravenous inoculation resulted in detectable colonies of tumor cells in animals receiving more than 1x10(6) cells. Our demonstrated ability to detect small numbers of tumor cells in living animals noninvasively suggests that therapies designed to treat minimal disease states, as occur early in the disease course and after elimination of the tumor mass, may be monitored using this approach. Moreover, it may be possible to monitor micrometastases and evaluate the molecular steps in the metastatic process. Spatiotemporal analyses of neoplasia will improve the predictability of animal models of human disease as study groups can be followed over time, and this method will accelerate development of novel therapeutic strategies.

Abstract

A method for studying bacteria that are attached to carcass surfaces would eliminate the need for exogenous sampling and would facilitate understanding the interaction of potential human food-borne pathogens with food animal tissue surfaces. We describe such a method in which we used a bioluminescent reporter strain of Escherichia coli O157:H7 that was constructed by transformation with plasmid pCGLS1, an expression vector that contains a complete bacterial luciferase (lux) operon. Beef carcass surface tissues were inoculated with the bioluminescent strain, and adherent bacteria were visualized in real time by using a sensitive photon-counting camera to obtain in situ images. The reporter strain was found to luminesce from the tissue surfaces whether it was inoculated as a suspension in buffer or as a suspension in a bovine fecal slurry. With this method, areas of tissues inoculated with the reporter strain could be studied without obtaining, excising, homogenizing, and culturing multiple samples from the tissue surface. Use of the complete lux operon as the bioluminescent reporter eliminated the need to add exogenous substrate. This allowed detection and quantitation of bacterial inocula and rapid evaluation of adherence of a potential human pathogen to tissue surfaces. Following simple water rinses of inoculated carcass tissues, the attachment duration varied with different carcass surface types. On average, the percent retention of bioluminescent signal from the reporter strain was higher on lean fascia-covered tissue (54%) than on adipose fascia-covered tissue (18%) following water washing of the tissues. Bioluminescence and culture-derived viable bacterial counts were highly correlated (r2 = 0.98). Real-time assessment of microbial attachment to this complex menstruum should facilitate evaluation of carcass decontamination procedures and mechanistic studies of microbial contamination of beef carcass tissues.

Abstract

Genetic analysis of human immunodeficiency virus type 1 (HIV-1) from cases of mother-to-infant transmission were analyzed in an effort to provide insights into the viral selection that may occur during transmission, as well as the timing and source of transmitted viruses. HIV-1 env genes obtained from seven mothers and their perinatally infected infants in Sweden were studied. Five envelope sequence clades (A to E) were found to be represented. We used a heteroduplex tracking assay (HTA) to assess the genetic relatedness between early viral isolates from the infants and serial maternal virus populations taken during pregnancy and at delivery. HTA findings were used to select for DNA sequence analysis maternal virus populations that were either closely or more distantly related to the infant virus. In each case, nucleotide sequence analysis confirmed the genetic relationships inferred by the HTA. Only maternal peripheral blood was sampled, and large sets of maternal specimens throughout pregnancy were generally not available. However, no consistent correlation was found to support the hypothesis that infant viruses should match blood-derived maternal virus genotypes found early in pregnancy if infants were found to be infected at birth or, conversely, that infant viruses should match blood-derived maternal virus genotypes found at delivery if infants were found to be infected only some time later.